Allosteric Modulators of the Mu Opioid Receptor

ABSTRACT

Disclosed herein are compounds, of the class of amine-bearing heterocycles, which act as positive allosteric modulators and silent allosteric modulators of the mu opioid receptor. These compounds are useful for the treatment of pain, drug addiction, and other CNS derived maladies that are controlled directly or indirectly by activation of the mu opioid receptor. Methods for making and using the allosteric modulators disclosed herein are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. Utility application Ser. No.16/048,051 filed on Jul. 27, 2018, which claims the benefit, under 35U.S.C. § 119(e)(1), of U.S. Provisional Application No. 62/605,106 filedon Jul. 31, 2017; the disclosures of which are hereby incorporated byreference, in their entireties, for all purposes.

FIELD

The present application is in the field of receptor biology; inparticular, opioid receptors and allosteric modulators thereof.

BACKGROUND

The superfamily of G protein-coupled receptors (GPCRs) comprises plasmamembrane spanning proteins that transduce signals via heterotrimeric Gproteins on the inner surface of the plasma membrane, leading tointracellular signaling cascades. Jacoby et al. (2006) ChemMedChem1:761-782. The cell surface location, tissue distribution, and diversityof these GPCRs make them ideal targets for drug intervention. It iswidely reported that roughly 30% of marketed drugs target specific GPCRactivity. Jacoby, E. B. (2006), supra; Overington et al. (2006) NatureReviews Drug. Disc. 5:993-996.

The Opioid Receptors (ORs) are members of the Class A family of GPCRs.As such, ORs mediate the actions of endogenous opioids (e.g.,endorphins) as well as the action of exogenous opioids such as morphineand morphine-like opiates, including most clinical analgesics. Four ORtypes are known to exist: the mu OR, the delta OR, the kappa OR and theL1OR; respectively abbreviated as MOR, DOR, KOR, ORL1. These OR subtypesappear to have overlapping functional mechanisms at a cellular level,share roughly 60% amino acid identity, and signal through the Gi/ofamily of heterotrimeric G proteins. The signaling pathways of opioidreceptors are well characterized. Stein & Machelska (2011)Pharmacological Reviews 63:860-881.

After binding of an orthosteric ligand, conformational changes in thereceptor allow intracellular coupling of heterotrimeric Gi/o proteins tothe C terminus of the receptor. At the Gα subunit, GTP replaces GDP anddissociation of the trimeric G protein complex into Gα and Gβγ subunitsensues. Subsequently, these subunits can inhibit adenylyl cyclases andthereby reduce cAMP production and/or directly interact with differention channels in the membrane.

The different OR types appear to share many functional mechanisms at thecellular level. The activation and/or inactivation of the ORs byendogenous or exogenous orthosteric opioids results in inhibition ofadenylyl cyclase, modulation of ion channel activity, andtranscriptional changes in the cell. Waldhoer et al. (2004) AnnualReview of Biochemistry 73:953-990. For example, activation of the muopioid receptors causes inhibition of adenylate cyclase (resulting inlower intracellular cAMP levels), and recruits β-arrestin to thereceptor. β-arrestin recruitment is a non-G-protein mediated signalingpathway through which many GPCRs (including the ORs) signal. Evidenceexists that β-arrestin is involved in receptor desensitization andinternalization/recycling. Whalen, E. R. (2011) Trends in MolecularMedicine 17:126-139; Shukla et al. (2011) Trends in Biomedical Sciences36:457-469.

ORs are key targets in the management of pain, with morphine and itsderivatives inducing pain relief by acting as full or partial receptoragonists. Pain relief (analgesia) is attributed to the actions ofopiates and opioids specifically at MOR. Matthes et al. (1996) Nature383:819-823; Manglik et al. (2012) Nature 485:321-326. When therapeuticdoses of morphine are given to patients with pain, the patients reportthat the pain is less intense, less discomforting, or entirely absent.However, in addition to providing relief of distress, the narrowtherapeutic window for morphine also clinically manifests a variety ofadverse side effects. Moreover, when a pain-relieving dose of morphineis administered to a pain-free individual, the experience is not alwayspleasant; nausea is common, and vomiting may also occur. In addition,drowsiness, inability to concentrate, difficulty in mentation, apathy,lessened physical activity, reduced visual acuity, and lethargy mayensue.

Two highly selective MOR agonists, endomorphin-1 (EM1) and endomorphin-2(EM2), have been isolated from bovine as well as human brains in largequantities and are believed to be the endogenous ligands for the MOR.Zadina et al. (1997) Nature 386:499-502; Hackler et al. (1993)Neuropeptides 24:159-164; Erchegyi et al. (1992) Peptides 13:623-631.Relative to other orthosteric agonists, EM1 and EM2 display anexceptionally high level of binding affinity and selectivity for MORover KOR and DOR. Zadina et al. (1994) Life Sciences 55:461-466. Thedifferential distribution of these peptides in various tissues has beenwidely studied with results indicating that EM1 is more denselydistributed throughout the brain; whereas EM2 is more prevalent in thespinal cord. Martin-Schild et al. (1999) J. Comp. Neurol. 405:450-471.Additionally, the presence of the endomorphins (EMs) and MOR has beenconfirmed in animal and human models of inflammatory and neuropathicpain. Troung et al. (2003) Ann. Neurol. 53:366-375; Straub et al. (2008)Arthritis and Rheumatism 58:456-466; Yang et al. (2014) PLOS ONE9(2):e89583; Stein et al. (1993) Lancet 342:321-324; Mousa et al. (2002)J. Neuroimmunol. 126:5-15; McDougall et al. (2004)Am. J. Physiol. Regul.Integr. Comp. Physiol. 286:R634-R641; Obara et al. (2004) NeuroscienceLetters 360:85-89. The antinociceptive actions of exogenouslyadministered EM1 and EM2 have been studied in a variety of animalmodels, as well as in humans. Soigner et al. (2000) Life Sciences67:907-912; Horvath et al. (1999) Life Sciences 65:2635-2641; Macdougallet al. (2004) J. Molec. Neurosci. 22:125-137; Horvath (2000)Pharmacology & Therapeutics 88:437-463; Przewlocka et al. (1999a) Eur.J. Pharmacol. 367:189-196; Przewlocka et al. (1999b) Ann NY Acad. Sci.897:154-164.

Opioid receptors have been extensively studied because of the needs for(1) better pain control and (2) reduction or elimination of adverse sideeffects. The side effects common to orthosteric ligands for the opioidreceptors include, in addition to those mentioned above, tolerance,respiratory suppression, constipation, allodynia, and dependence.Waldhoer et al., supra; McNicol et al. (2003) J. Pain 4:231-256. Indeed,recent determinations of the therapeutic indices for commonly usedopioids led to the conclusion that systemic side effects are to beexpected for all of them (Kuo et al. (2015) British J. Pharmacol.172:532-548), leading some to conclude that the side effects aremechanism-based. Alternatively, the side effects of opioid use could beattributed to the signal bias that these orthosteric ligands induceand/or to the presence of receptors and/or their ligands in tissues thatare not experiencing pain. Kenakin (2015a) Trends Pharmacol. Sci.36:705-706; Stein & Machelska, supra.

To overcome the side effects associated with traditional OR orthostericagonists and partial agonists, drug discovery efforts have focused on(1) developing selective orthosteric ligands which display OR subtypeselectivity either as full agonists, partial agonists or when used incombination therapy (Davis (2012) Exp. Opin. Drug Discov. 7:165-178;Dietis (2009) Br. J. Anaesth. 103:38-49); (2) physiologicalcompartmentalization of orthosteric ligands (Spahn et al. (2017) Science355:966-969); and (3) inducing selective signal bias of orthostericligands (Soergel et al. (2014) Pain 155:1829-1835; Chen et al. (2013) J.Medicinal Chem. 56:8019-8031). These diverse approaches have in common astrategy in which the orthosteric ligand binding domain is the onlyentity being probed and/or modified. Given the current national epidemicof orthosteric opioid agonist abuse, a novel approach to pain mitigationwould provide a welcome benefit to society. Lauren & Rossen (2016) DrugPoisoning Mortality: United States, 2002-2014. Atlanta: National Centerfor Health Statistics, Centers for Disease Control and Prevention.

Allosteric modulators of opioid receptors provide an alternativestrategy for pain mitigation. An allosteric modulator has no intrinsicagonist or antagonist activity toward a receptor but, in the presence ofan orthosteric agonist, can further increase the activity of thereceptor beyond that induced by the orthosteric agonist (a positiveallosteric modulator, or PAM) or decrease receptor activity below thatwhich would normally be induced by the orthosteric agonist (a negativeallosteric modulator, or NAM). Thus, the use of positive allostericmodulators of the μ-opioid receptor (MOR PAMS or MOR-PAMs), when used inconjunction with exogenous opioids, would allow lower doses of opioid tobe administered, thereby lessening side effects and potential for abuse.

In addition, because endogenous MOR agonists are not distributedthroughout the entire body, but tend to be released in areasexperiencing pain, the use of a MOR PAM as an analgesic (in the absenceof an exogenous opioid) also has the advantage that the body of thesubject is not being flooded with an exogenous opioid. Rather, theMOR-PAM activates the receptor only in the regions of the body thatalready contain endogenous receptor agonists; thereby potentiating theactivity of the agonist and activating the receptor only in the regionswhere necessary for pain reduction.

The naturally-occurring hallucinogen Salvinorin A has been observed tobehave, in vitro, as an allosteric modulator of the MOR. Rothman (2007)J. Pharmacol. Exp. Therapeutics 320:801-810. However, further use ofthis compound for in vivo mechanistic testing and/or therapeutic use isunlikely due to its inherent polypharmacology.

Thiazolidine-based allosteric modulators of the MOR have also beendescribed and tested in vitro. WO 2014/107344; Burford et al. (2011)Biochemical Pharmacol. 81:691-702; Burford et al. (2013) Proc. Natl.Acad. Sci. USA 110:10830-10835; Burford et al. (2015) Br. J. Pharmacol.172:277-286; Livingston et al. (2014) Proc. Natl. Acad. Sci. USA111:18369-18374. However, in vitro analyses of these compounds haverevealed that they are unlikely to be useful for in vivo studies androutine therapeutic use, because of the difficulty of their synthesis,poor potency and metabolic instability. Some of these compounds alsopossess pure agonist activity in addition to PAM activity in certainassays (i.e., they act as “ago-PAMs”) and therefore lack the benefit ofbeing able to amplify endogenous analgesic mechanisms in a temporally-and spatially-limited fashion. (Bisignano et al. (2015) J. Chem. Inf.Model 55:1836-1843; Rockwell & Alt (2017) “Positive AllostericModulators of Opioid Receptors” in RSC Drug Discovery Series No. 56:Allosterism in Drug Discovery, ed. D. Doller, Royal Society ofChemistry, Chapter 9, pp. 194-219; Livingston et al., supra.

Modification of these thiazolidine-based MOR PAMs, guided bystructure/activity relationship (SAR)-based lead optimization, andattendant pharmacophore modeling, have been conducted. (Bisignano et al.(2015) J. Chem. Inf. Model 55:1836-1843; Bartuzi et al. (2016) J. Chem.Inf. Model 56:563-570. These efforts have also not yielded potent orselective compounds viable for in vivo testing. In addition, theSAR-based derivatives are somewhat non-specific, showing some PAMactivity at the delta opioid receptor as well. Rockwell & Alt, supra.

For all of the foregoing reasons, there is a need for new compounds thatinduce OR signaling and analgesia, but have reduced side effects, andthat are suitable for in vivo testing and administration. There is acontinuing need for new analgesics that can incorporate the profoundbeneficial effects of opioids without the concomitant side effects; andfor compounds that can potentiate the temporally- andspatially-restricted activity of endogenous opioids.

SUMMARY

Disclosed herein are compositions that act as positive allostericmodulators (PAMs) of mu opioid receptor-mediated signal transduction,methods for their synthesis, and methods for their use in providinganalgesia with minimal side effects. For inducing analgesia, PAMs can beadministered by themselves to augment the activity of endogenous opioidssuch as the endomorphins. Alternatively, PAMs can be administered incombination with an exogenous opioid such as, for example, morphine,oxycodone or fentanyl. In these embodiments, a MOR PAM can beadministered at the same time (i.e., together with) an exogenous opioid,or the MOR PAM and the exogenous opioid can be administered at differenttimes. If administered at different times, administration or the MOR PAMcan precede administration of the exogenous opioid, or administration ofthe exogenous opioid can precede administration of the MOR PAM.

In certain embodiments, compounds disclosed herein act as positiveallosteric modulators (PAMs) of the mu opioid receptor (MOR). By itself,a PAM bound to the receptor has no effect on receptor activity. However,when the MOR is bound by both a PAM as disclosed herein and anorthosteric ligand (endogenous or exogenous); the signaling activity ofthe MOR is greater than when it is bound by an orthosteric ligand alone.Endogenous orthosteric MOR ligands include endomorphin-1 (EM1);endomorphin-2 (EM2), the enkephalins (e.g., Leu-Enk and Met-Enk, whichalso bind the delta- and kappa-opioid receptors) and β-endorphin (whichalso bind the delta- and kappa-opioid receptors). Exogenous orthostericMOR ligands include, for example, morphine, morphine-6-glucuronide,fentanyl, oxycontin, oxycodone, hydrocodone, DAMGO, herkinorn,loperamide, buprenorphine, etorphine, methadone, naloxone, Oxymorphonehydrazine, [D-penicillamine 2,5]-enkephalin (DPDE), Hydromorphone,Dihydromorphine, Codeine, Oxymorphol, and Oxymorphone.

In certain embodiments, the compounds disclosed herein potentiate theactivity of endomorphin-1 (EM1) on the mu opioid receptor. In additionalembodiments, the compounds disclosed herein potentiate the activity ofendomorphin-2 (EM2) on the mu opioid receptor.

MOR signaling can be measured by methods that are known in the artincluding, but not limited to, recruitment of beta-arrestin to thereceptor, inhibition of adenylate cyclase activity (resulting inlowering of intracellular cAMP levels in the receptor cell),phosphorylation of extracellular signal-related kinases 1 and 2 (ERK1/2;also known as mitogen-activated protein kinase, MAPK) and G-proteinactivation (which involves a conformational change in the receptor thatallows exchange of bound GDP for GTP).

Thus, in certain embodiments, MOR PAMs as disclosed herein augment oneor more of beta-arrestin-1/2 recruitment, inhibition of adenylatecyclase activity, phosphorylation of ERK1/2 and G-protein activation.

In certain embodiments, MOR PAMs as disclosed herein selectivelymodulate the activity of the mu opioid receptor, as compared to thedelta, kappa, or ORL1 opioid receptors. In additional embodiments,MOR-PAMs as disclosed herein specifically potentiate the activity ofendomorphin-1 on the MOR, compared to other orthosteric ligands of theMOR. In additional embodiments, MOR-PAMs as disclosed hereinspecifically potentiate the activity of endomorphin-2 on the MOR,compared to other orthosteric ligands of the MOR.

Certain of the PAMs disclosed herein exhibit selective signal bias onG-protein-mediated downstream signaling activities; i.e., they affect aparticular downstream process (e.g., cAMP signaling) more strongly thanother downstream processes (e.g., β-arrestin recruitment, GTP/GDPexchange, ERK phosphorylation). Selective signal bias is beneficial, forexample, for cases in which a particular downstream signaling systemcontributes to the side effects of an opioid rather than to itsanalgesic effect (e.g., β-arrestin recruitment).

Also provided are compositions that act as silent allosteric modulators(SAMs) of mu opioid receptor-mediated signal transduction, methods fortheir synthesis, and methods for their use. Thus, in certainembodiments, compounds disclosed herein act as silent allostericmodulators (SAMs) of the mu opioid receptor (MOR). A SAM binds to a siteon the receptor that is identical to, or overlapping with, the sitebound by a PAM; however, binding of the SAM has no effect on theactivity (agonistic or antagonistic) of the orthosteric ligand. Althougha SAM cannot directly affect the activity of an orthosteric ligand, itcan modulate the effect of a PAM on the orthosteric ligand, by competingwith the PAM for binding to the receptor. Thus, for example, a SAM canbe used, in combination with a PAM, to fine-tune the modulatory effectof the PAM.

Accordingly, in additional embodiments for the use of MOR PAMs, the MORPAM is administered together with a MOR SAM and optionally an exogenousopioid. Administration of the MOR PAM and the MOR SAM can be conductedat the same time or at different times. If administered at differenttimes, administration of the MOR PAM can precede administration of theMOR SAM, or administration of the MOR SAM can precede administration ofthe MOR PAM. Administration of the MOR PAM and/or the MOR SAM can eitherprecede or follow administration of the exogenous opioid, oradministration of the MOR PAM and/or the MOR SAM can occursimultaneously with the administration of the exogenous opioid.

Also provided are methods of treating pain in a subject in need thereofby administering to the subject a MOR PAM as disclosed herein. Inadditional embodiments, methods of treating pain in a subject compriseadministering to the subject a MOR PAM as disclosed herein inconjunction with a MOR SAM as disclosed herein. The MOR PAM and/or MORPAM/MOR SAM combination can be administered before or during the onsetof pain and, upon the release of endogenous opioids (e.g., EM1), thepresence of the MOR PAM in the subject increases the intensity and/orduration of the native pain-relieving mechanism.

In additional embodiments, methods of treating pain in a subjectcomprise administering to the subject an orthosteric ligand of an opioidreceptor in conjunction with a MOR PAM as disclosed herein. In furtherembodiments, methods of treating pain in a subject compriseadministering to the subject an orthosteric ligand of an opioid receptorin conjunction with both a PAM as disclosed herein and a SAM asdisclosed herein. In certain embodiments, the opioid receptor is theMOR. In additional embodiments, the orthosteric agonist is, for example,morphine, oxycodone or fentanyl.

Also provided are methods for inducing analgesia in a subject in needthereof, by administering to the subject a MOR PAM as disclosed herein.In additional embodiments, methods of inducing analgesia in a subjectcomprise administering to the subject a MOR PAM as disclosed herein inconjunction with a MOR SAM as disclosed herein. The MOR PAM and/or MORPAM/MOR SAM combination can be administered before or during the onsetof pain and, upon the release of endogenous opioids (e.g., EM1), thepresence of the MOR PAM in the subject increases the intensity and/orduration of native analgesic mechanisms.

In additional embodiments, methods of inducing analgesia in a subjectcomprise administering to the subject an orthosteric ligand of an opioidreceptor in conjunction with a MOR PAM as disclosed herein. In furtherembodiments, methods of inducing analgesia in a subject compriseadministering to the subject an orthosteric ligand of an opioid receptorin conjunction with both a PAM as disclosed herein and a SAM asdisclosed herein. In certain embodiments, the opioid receptor is theMOR. In additional embodiments, the orthosteric agonist is, for example,morphine, oxycodone or fentanyl.

Also provided are methods for reducing nociception in a subject in needthereof, by administering to the subject a MOR PAM as disclosed herein.In additional embodiments, methods of reducing nociception in a subjectcomprise administering to the subject a MOR PAM as disclosed herein inconjunction with a MOR SAM as disclosed herein. The MOR PAM and/or MORPAMIMOR SAM combination can be administered before or during the onsetof pain and, upon the release of endogenous opioids (e.g., EM1), thepresence of the MOR PAM in the subject reduces the intensity and/orduration of nociception.

In additional embodiments, methods of reducing nociception in a subjectcomprise administering to the subject an orthosteric ligand of an opioidreceptor in conjunction with a MOR PAM as disclosed herein. In furtherembodiments, methods of reducing nociception in a subject compriseadministering to the subject an orthosteric ligand of an opioid receptorin conjunction with both a PAM as disclosed herein and a SAM asdisclosed herein. In certain embodiments, the opioid receptor is theMOR. In additional embodiments, the orthosteric agonist is, for example,morphine, oxycodone or fentanyl.

In certain embodiments for the use of a MOR-PAM (optionally incombination with a MOR-SAM) to treat pain, induce analgesia and/orreduce nociception; the MOR-PAM (optionally in combination with aMOR-SAM) is administered together with exogenously-administered (e.g.,synthetic) EM1 or EM2 (e.g., Cyt-1010, Cytogel Pharma, Darien CT). See,for example, U.S. Pat. Nos. 5,885,958; 6,303,578 and 8,716,436.

Also provided are methods for potentiating the effect of an exogenousopioid administered to a subject by co-administering, with the exogenousopioid, a MOR PAM as disclosed herein and/or a MOR SAM as disclosedherein. In certain embodiments, a reduced dose of the exogenous opioidis co-administered with the PAM or PAMISAM combination. The MOR PAM canbe administered prior to, at the same time as, or subsequent to,administration of the exogenous opioid. The MOR SAM can be administeredprior to, at the same time as, or subsequent to, administration of theexogenous opioid and/or the MOR PAM.

In additional embodiments, methods for reducing the side effects of anopioid are provided. In these methods, the opioid (e.g., morphine,oxycodone, fentantyl, etc.) is administered at a lower-than-normal dose(i.e., a sub-therapeutic dose), in combination with a MOR PAM asdisclosed herein and/or a MOR PAM as disclosed herein and a MOR SAM asdisclosed herein. The MOR PAM can be administered prior to, at the sametime as, or subsequent to, administration of the exogenous opioid. TheMOR SAM can be administered prior to, at the same time as, or subsequentto, administration of the exogenous opioid and/or the MOR PAM. Incertain embodiments, the MOR PAM possesses selective signal bias; e.g.,toward adenylyl cyclase inhibition and away from β-arrestin recruitment.

The MOR PAM compounds disclosed herein can be used to treat acute orchronic pain arising from any type of disorder including, but notlimited to, inflammation, blunt force, cancer, neuropathy, burns,surgery, hormonal or endocrine imbalances, and viral, fungal and/orbacterial insults. A partial but not exhaustive list of therapeutic usesfor such compounds includes treatment of pain, immune dysfunction,inflammation, esophageal reflux, neurological and psychiatricconditions, medicaments for drug and alcohol abuse, agents for treatinggastritis and diarrhea, cardiovascular agents and/or agents for thetreatment of respiratory diseases and cough.

The MOR PAM compounds disclosed herein can also be used to treatnon-pain disorders arising from conditions of known and unknown etiologythat have physiological connections to endogenous circulating endorphinlevels. These include but are not limited to any type of hormonal orendocrine imbalances which can affect an individual. A partial but notexhaustive list of therapeutic uses for such compounds includestreatment of anxiety, depression, stress, urological and reproductiveconditions, and sexual dysfunction.

In the methods of use disclosed herein, the MOR PAM compounds can beadministered to a subject by any means known in the art including, forexample, injection, transdermal, parenteral, intravenous,intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital,ophthalmic, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intracerebroventricular, intrathecal,intranasal, aerosol, by suppositories, or by oral administration.

Also provided are pharmaceutical compositions comprising a MOR PAMcompound as disclosed herein and a pharmaceutically acceptableexcipient.

Also provided are MOR PAM compounds as disclosed herein for use inreducing pain, inducing analgesia, or reducing nociception in a subject.

Also provided are uses of MOR PAM compounds as disclosed herein inmethods for reducing pain, inducing analgesia, or reducing nociceptionin a subject.

Also provided are uses of MOR PAM compounds as disclosed herein in themanufacture of a medicament for reducing pain, inducing analgesia, orreducing nociception in a subject.

Also provided are kits for use in reducing pain, inducing analgesia, orreducing nociception in a subject, wherein the kits comprise one or moreMOR PAM compounds as disclosed herein, optionally in combination with apharmaceutically acceptable excipient and/or packaging or container(s)and/or instruction for use.

Also provided are pharmaceutical compositions comprising a MOR SAMcompound as disclosed herein and a pharmaceutically acceptableexcipient.

Also provided are MOR SAM compounds as disclosed herein for use inreducing pain, inducing analgesia, or reducing nociception in a subject.

Also provided are uses of MOR SAM compounds as disclosed herein inmethods for reducing pain, inducing analgesia, or reducing nociceptionin a subject.

Also provided are uses of MOR SAM compounds as disclosed herein in themanufacture of a medicament for reducing pain, inducing analgesia, orreducing nociception in a subject.

Also provided are kits for use in reducing pain, inducing analgesia, orreducing nociception in a subject, wherein the kits comprise one or moreMOR SAM compounds as disclosed herein, optionally in combination with apharmaceutically acceptable excipient and/or packaging or container(s)and/or instruction for use.

Also provided are methods for making positive allosteric modulators andsilent allosteric modulators of opioid receptors. These methods aredescribed elsewhere in this specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a concentration response curve for Compound 44 in abeta-arrestin recruitment assay using the MOR and an EC₂₀ endomorphin-1concentration of 4.5 nM. The compound exhibits moderate positiveallosteric activity for β-arrestin recruitment.

FIG. 1B shows a concentration response curve for Compound 44 in a cAMPsignaling assay using the MOR and an EC₂₀ endomorphin-1 concentration of0.45 nM. The compound exhibits moderate positive allosteric activity forcAMP signaling.

FIG. 2A shows a concentration response curve for Compound 219 in abeta-arrestin recruitment assay using the MOR and an EC₂₀ endomorphin-1concentration of 4.5 nM. The compound exhibits low positive allostericactivity for β-arrestin recruitment.

FIG. 2B shows a concentration response curve for Compound 219 in a cAMPsignaling assay using the MOR and an EC₂₀ endomorphin-1 concentration of0.45 nM. The compound exhibits high positive allosteric activity forcAMP signaling.

FIG. 3A shows a concentration response curve for Compound 14 in abeta-arrestin recruitment assay using the MOR and an EC₂₀ endomorphin-1concentration of 4.5 nM. The compound exhibits silent allostericactivity for β-arrestin recruitment.

FIG. 3B shows a concentration response curve for Compound 14 in a cAMPsignaling assay using the MOR and an EC₂₀ endomorphin-1 concentration of0.45 nM. The compound exhibits silent allosteric activity for cAMPsignaling.

FIG. 4A shows a concentration response curve for Compound 216 in abeta-arrestin recruitment assay using the MOR and an EC₂₀ endomorphin-1concentration of 4.5 nM. The compound exhibits high positive allostericactivity for β-arrestin recruitment.

FIG. 4B shows a concentration response curve for Compound 216 in a cAMPsignaling assay using the MOR and 4 an EC₂₀ endomorphin-1 concentrationof 0.45 nM. The compound exhibits silent allosteric activity for cAMPsignaling.

FIG. 5A shows a concentration response curve for Compound 2 in abeta-arrestin recruitment assay using the MOR and an EC₂₀ endomorphin-1concentration of 4.5 nM. The compound exhibits high positive allostericactivity for β-arrestin recruitment.

FIG. 5B shows a concentration response curve for Compound 2 in a cAMPsignaling assay using the MOR and an EC₂₀ endomorphin-1 concentration of0.45 nM. The compound exhibits high positive allosteric activity forcAMP signaling.

FIG. 6A is a concentration response curve for Compound 108 in abeta-arrestin recruitment assay using the MOR and an EC₂₀ concentrationof 4.5 nM endomorphin-1. The compound exhibits a moderate positiveallosteric effect on EM1 agonism of the MOR.

FIG. 6B is a concentration response curve for Compound 108 in a cAMPsignaling assay using the MOR and an EC₂₀ concentration of 0.45 nMendomorphin-1. The compound exhibits a moderate positive allostericeffect on EM1 agonism of the MOR.

FIG. 6C is a concentration response curve for Compound 177 in abeta-arrestin recruitment assay using the MOR and an EC₂₀ concentrationof 4.5 nM endomorphin-1. The compound exhibits a moderate positiveallosteric effect on EM1 agonism of the MOR.

FIG. 6D is a concentration response curve for Compound 177 in a cAMPsignaling assay using the MOR and an EC₂₀ concentration of 0.45 nMendomorphin-1. The compound exhibits a moderate positive allostericeffect on EM1 agonism of the MOR.

FIG. 6E is a concentration response curve for Compound 6 in abeta-arrestin recruitment assay using the MOR and an EC₂₀ concentrationof 4.5 nM endomorphin-1. The compound exhibits a moderate positiveallosteric effect on EM1 agonism of the MOR.

FIG. 6F is a concentration response curve for Compound 6 in a cAMPsignaling assay using the MOR and an EC₂₀ concentration of 0.45 nMendomorphin-1. The compound exhibits a moderate positive allostericeffect on EM1 agonism of the MOR.

FIG. 6G is a concentration response curve for Compound 110 in abeta-arrestin recruitment assay using the MOR and an EC₂₀ concentrationof 4.5 nM endomorphin-1. The compound exhibits a moderate positiveallosteric effect on EM1 agonism of the MOR.

FIG. 6H is a concentration response curve for Compound 110 in a cAMPsignaling assay using the MOR and an EC₂₀ concentration of 0.45 nMendomorphin-1. The compound exhibits a moderate positive allostericeffect on EM1 agonism of the MOR.

FIG. 6I is a concentration response curve for Compound 109 in abeta-arrestin recruitment assay using the MOR and an EC₂₀ concentrationof 4.5 nM endomorphin-1. The compound exhibits a moderate positiveallosteric effect on EM1 agonism of the MOR.

FIG. 6J is a concentration response curve for Compound 109 in a cAMPsignaling assay using the MOR and an EC₂₀ concentration of 0.45 nMendomorphin-1. The compound exhibits a moderate positive allostericeffect on EM1 agonism of the MOR.

FIG. 7A shows concentration response curves (CRCs) for the effect ofcompound 9 on oxycodone-induced β-arrestin recruitment.

FIG. 7B shows concentration response curves (CRCs) for the effect ofcompound 9 on oxycodone-induced adenylyl cyclase inhibition.

FIG. 8A shows the effect of increasing concentration of compound 60 onβ-arrestin recruitment by the delta opioid receptor (DOR) induced by anEC₂₀ concentration of the DOR agonist d-Ala², D-Leu⁵-enkephalin (DADLE).

FIG. 8B shows the effect of increasing concentrations of DADLE onβ-arrestin recruitment by the delta opioid receptor.

FIG. 9A shows the effect of increasing concentration of compound 60 onβ-arrestin recruitment by the kappa opioid receptor (KOR) induced by anEC₂₀ concentration of the KOR agonist Dynorphin A.

FIG. 9B shows the effect of increasing concentrations of Dynorphin A onβ-arrestin recruitment by the kappa opioid receptor.

FIG. 10A is a concentration response curve showing the effect ofincreasing concentrations of compound 6 on oxycodone-induced β-arrestinrecruitment by the MOR.

FIG. 10B is a concentration response curve showing the effect ofincreasing concentrations of compound 6 on oxycodone-induced adenylylcyclase inhibition by the MOR.

FIG. 11A shows a time-course of plasma concentration of compound 9 inCD-1 mice (n=2) after either subcutaneous (SC) or intraperitoneal (IP)administration of 15 mg/kg of compound 9.

FIG. 11B shows mean (n=2) concentrations of compound 9 in plasma, brainand cerebrospinal fluid (CSF); one hour after administration of 15 mg/kgof compound 9 to CD-1 mice by subcutaneous (SC) or intraparietal (IP)injection. Concentrations of compound 9 were measured by massspectroscopy.

FIG. 11C shows mean (n=3) concentrations of compound 9 in plasma, brainand spinal cord of ICR rats; at different times (0.5, 1.0 and 1.5 hours)after subcutaneous injection of 15 mg/kg of the compound. Concentrationsof compound 9 were measured by mass spectroscopy.

FIG. 12A shows effect of compound 9 on the tail-flick response in CD1mice in the absence of added exogenous EM-1 (triangles). The effect ofmorphine is also shown (squares). Tail flick latency was measured 30minutes after subcutaneous introduction of either compound 9 ormorphine. Injection of an equal volume of vehicle was used as a negativecontrol (circles).

FIG. 12B shows a time-course of the effect of added exogenousendomorphin-1 (Endo) on the tail-flick response in ICR rats. Three dosesof EM-1 were introduced by intrathecal injection: 1 μg (diamonds), 3 μg(triangles) and 10 μg (squares); and an equal volume of saline (x) wasinjected as a negative control.

FIG. 12C shows a time course of the effect of two concentrations ofsubcutaneously administered compound 9 (15 and 30 mg/kg) on theantinociceptive effect of a minimally efficacious dose of intrathecallyadministered exogenous EM1 (3 μg, triangles) in the tail-flick assay.Concentrations of compound 9 administered were 15 mg/kg (+) and 30 mg/kg(circles). Also shown are results after administration 10 μg of EM1(squares) and saline.

FIG. 12D shows a time course of the effect of two concentrations ofsubcutaneously administered compound 9 (15 mg/kg, + symbol, and 30mg/kg, circles) on the antinociceptive effect of a sub-effecaceous doseof intrathecally administered exogenous EM1 (1 μg, diamonds) in thetail-flick assay. Effects of administration of an equal volume ofsaline, and of administration of 3 μg (triangles) and 10 μg (squares) ofEM1 are shown for comparison.

FIG. 13 shows effects of compound 9 (at 15, 30 and 60 mg/kg body weight)on gastrointestinal transit of charcoal through rat small intestine.Effects of no compound (vehicle) and morphine were also determined.

FIG. 14 shows effects of compound 9 (at 15, 30 and 60 mg/kg body weight)on amount of charcoal remaining in rat stomach 20 minutes afteradministration by lavage. Effects of no compound (vehicle) and morphinewere also determined.

FIG. 15 shows oxygen saturation, measured by pulse oximetry, in ratstreated with compound 9 (at 15, 30 and 60 mg/kg body weight). Also shownare effects of morphine and vehicle.

DETAILED DESCRIPTION

Practice of the present disclosure employs, unless otherwise indicated,standard methods and conventional techniques in the fields of organicchemistry, synthetic chemistry, biochemistry, pharmacology, cellbiology, toxicology, molecular biology, cell culture, recombinant DNAand related fields as are within the skill of the art. Such techniquesare described in the literature and thereby available to those of skillin the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thecompositions and compounds described herein, suitable methods andmaterials are described herein.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entireties. Inaddition, the materials, methods, and examples are illustrative only,and not intended to be limiting. Other features and advantages of thecompositions and compounds described herein will be apparent from thefollowing detailed description and claims.

Disclosed herein are compounds that act as of MOR PAMs and MOR SAMs. TheMOR PAMs and MOR SAMs disclosed herein function with respect to bothendogenous MOR ligands (e.g., EM-1 and EM-2) and a variety ofnon-endogenous orthosteric ligands (e.g., morphine, oxycodone, fentanyl,loperamide). Certain of the MOR PAMs and MOR SAMs disclosed hereinexhibit selective signal bias; that is, they influence certainligand-induced MOR downstream signaling activities more strongly thanothers. For example, certain MOR PAMs have a stronger effect on adenylylcyclase inhibition than on β-arrestin recruitment. Since β-arrestinrecruitment is believed to contribute more strongly to the side effectsexperienced with the use of non-endogenous opioids, MOR PAMs withselective signal bias toward adenylyl cyclase inhibition (and away fromβ-arrestin recruitment) provide improved therapeutics for use inanalgesia and pain management.

In the absence of EM1 (an endogenous MOR ligand), the MOR PAMs disclosedherein show neither agonist nor antagonist activity in assays for MORactivation (described elsewhere herein), thus demonstrating a pure PAMmechanism. MOR PAMs, as disclosed herein, demonstrated a left shift in aSchild-regression analysis of EM1, demonstrating that they have animpact on alpha, beta and tau-B values for the endogenous ligandendomorphin-1 Kenakin & Willilams (2014) Biochem. Pharmacol. 87:40-63.Certain of the MOR PAMs disclosed herein were demonstrated to have noPAM activity against DOR and KOR subtypes, thus demonstrating a level ofselectivity difficult to achieve with orthosteric ligands.

The MOR PAMs disclosed herein are selective for the endogenousorthosteric ligands EM1 and EM2, but some also retain activity fornon-natural orthosteric ligands such as oxycodone. For example, certainof the MOR PAMS disclosed herein demonstrated a left shift in aSchild-regression analysis for the non-endogenous ligand oxycodone,demonstrating that they are having an impact on alpha, beta and tau-Bvalues. This unexpected result demonstrates the versatility of the MORPAM approach for identifying therapeutically relevant compounds. Unlikepreviously identified MOR PAMs, compounds described herein show animalpharmacokinetic properties that demonstrate suitability for in vivotesting. Finally, the compounds described herein affect the efficacy andduration of analgesic effect in an animal model of acute pain.

Use of the MOR PAMs described herein for pain management combines theconcepts of allosteric ligands and selective signal bias. Allostericligands for a GPCR bind to a site on the receptor that is distinct fromthe orthosteric site to which endogenous ligands bind. Burford et al.(2011) supra; Kenakin (2009) Trends Pharmacol Sci. 30:460-469. Anallosteric modulator (AM) can exhibit a range of activities at thetarget protein by affecting a multitude of processes; such as, but notlimited to, the binding affinity of ligands at the orthosteric site, thedissociative off rate of ligands that bind to the orthosteric site, theform of the 7 transmembrane domain (7TM) protein available for bindingorthosteric ligands, and/or the form of the 7TM protein which transferssignals from the extracellular to the intracellular compartment.Positive allosteric modulators (PAMs) can increase the affinity (β)and/or the efficacy (α) of agonists and there are therapeutic settingsin which it is advantageous to distinguish between these mechanisms ofaction. For instance, in cases in which a failing physiological systemmust be revitalized, only efficacy effects (α>1) will be beneficial;however, for cases in which enhancement of a normally functioning systemis required, either α or β effects will suffice and, in general,molecules can be characterized with the αβ product parameter. Thepositive allosteric modulators (PAMs) described herein have no intrinsicagonist activity, but when bound to the receptor in concert with anorthosteric ligand (endogenous or otherwise), they enhance the bindingaffinity or efficacy (or both) of an orthosteric agonist or partialagonist. The silent allosteric modulators (SAMs) described have nointrinsic agonist activity, but when they bind to the receptor inconcert with an orthosteric ligand (endogenous or otherwise) they caninterfere with the binding of PAMS; thereby acting as competitiveantagonists by blocking the activity of the PAM.

Allosteric ligands have the potential to exhibit greater selectivity,compared with orthosteric ligands, between subtypes of GPCRs in the samefamily. This has been demonstrated for some GPCRs including metabotropicglutamate receptors, adenosine receptors, and muscarinic receptors.Birdsall (2005) Mini Reviews in Medicinal Chemistry 5:523-543; Bruns &Fergus (1990) Mol. Pharmacol. 38:939-949; Conn et al. (2009) TrendsPharmacol. Sci. 30:148-155; Gao et al. (2005) Mini Reviews in MedicinalChemistry 5:545-553; Harrington et al. (2010) Bioorganic & MedicinalChemistry Letters 20:5544-5547. This increased selectivity ishypothesized to be based on the evolutionary constraint placed on theorthosteric site between closely related receptor subtypes that bind thesame endogenous orthosteric ligand. This evolutionary constraint may notbe required for allosteric sites. Positive allosteric modulators of thecalcium-sensing receptor have been identified and shown to be clinicallyrelevant in the maintenance of cellular calcium concentrations.Harrington et al., supra.

There is data that indicates that the continuous availability ofendogenous opioids in inflamed tissue increases recycling and preservessignaling of MOR in sensory neurons. This is thought to counteract thedevelopment of peripheral opioid tolerance. Consequently, the use ofperipherally acting opioids for the prolonged treatment of inflammatorypain (optionally in combination with MOR-PAMs and/or MOR SAMs asdescribed herein) may not necessarily lead to opioid tolerance. Zollneret al. (2008) J. Clin. Investig. 118:1065-1073.

While highly selective orthosteric agonist ligands exist for the opioidreceptor subtypes, the PAMs described herein provide additionaladvantages. PAMs, unlike allosteric agonists, may have no effect whenthey bind to the receptor in the absence of an orthosteric agonist.Therefore, the modulation occurs only when an orthosteric agonist isbound to the receptor. In vivo, this leads to preservation of thetemporal and spatial characteristics of cell signaling and analgesicresponses mediated by endogenous orthosteric agonists; which isimportant, especially for signaling in the complex neuronal networks inthe brain and enteric nervous system. Additionally, by preserving thetemporal and spatial aspects of native receptor signaling, the use ofPAMs helps avoid receptor down-regulation and other compensatorymechanisms that are triggered by sustained receptor activation producedby exogenous orthosteric agonists. Zollner et al., supra.

In contrast to the use of a MOR PAM to potentiate the temporal andspatially-limited activity of endogenous orthosteric agonists asdescribed above; exogenous orthosteric agonists have the capability toactivate desired and undesired receptors in desired and undesiredtissues for an extended period of time, thereby resulting in off-targeteffects.

In response to pain, both humans and rodents produce ORs at the site ofinjury with concomitant endogenous opioid ligand trafficking occurringthrough a number of different mechanisms. Przewlocki et al. (1992)Neuroscience 48:491-500; Rittner et al. (2001) Anesthesiology95:500-508; Mousa et al., supra; Martin-Schild et al., supra; Li et al.(2005) Arthritis and Rheumatism 52: 3210-3219; Straub, et al., supra.Long-term systemic dosing with opiates is known, in some cases, to leadto the development of tolerance and dependence; as well as other acutereceptor-mediated side-effects such as respiratory suppression,constipation and allodynia. Waldhoer et al., supra; McNicol et al.,supra.

Thus, PAMs as disclosed herein, which have probe dependency forendogenous opioid agonists, take advantage of the body's natural painresponse mechanisms, acting primarily when the body is in pain, and thusretaining the temporal benefits of endogenous pain relief. By contrast,the use of traditional agonist ligands results in receptor activationfor long time periods (based on the dosing regime), often resulting inadverse effects, such as desensitization of the receptor response orreceptor-mediated side-effects caused by long-term stimulation. The useof MOR PAMS, as disclosed herein, reduces this long-term receptorstimulation, by increasing the efficacy of the agonist effect of theorthosteric ligand. Consequently, opioid receptor PAMs are expected toproduce less tolerance and dependence than exogenous orthostericagonists (Zollner et al., supra); leading to reduced abuse potential ofPAMs compared to that of orthosteric ligands

Another advantage of the PAMs disclosed herein is their ability toincrease the potency of an orthosteric agonist (as manifested by aleft-shift in the concentration response curve) by a finite amount.Burford et al. (2015), supra. This finite potency shift allows thedesign of PAMs that cannot exceed a required level of effect; therebyimproving safety.

It has been well documented in both humans (Stein et al. (1993), supra;Troung et al., supra) and non-humans (Borzsei (2008) Neuroscience152:82-88) that there is a temporally-regulated production of opioidreceptors in peripheral tissue in response to pain. Additionally, it isdocumented that endogenous ligands of the opioid receptors are releasedupon painful stimuli in animal models. Mousa et al., supra;Martin-Schild et al., supra; Yang et al., supra. The discovery anddevelopment of the PAMs disclosed herein which augment the efficacy,potency, duration of action, etc. of native and non-native ligands forthe ORs represents a significant advance in the state of modern drugdiscovery and pain management.

Assays for Ligand Binding and Opioid Receptor Activity

A “natural ligand-induced activity” as used herein, refers to activationof the MOR by endomorphin-1, enodomophin-2 or other endogenouslyproduced peptides. A “non-natural ligand-induced activity” as usedherein, refers to activation of the MOR by morphine, oxycodone, fentanylor other non-endogenously produced opiates. Activity can be assessedusing any number of endpoints to measure OR activity. Generally, assaysfor testing compounds that modulate MOR-mediated signal transduction,either by natural or non-natural ligand-induced activity, include thedetermination of any parameter that is directly or indirectly under theinfluence of an OR, e.g., a functional, physical, or chemical effect.Examples of functional effects include GTP/GDP exchange at the receptor,phosphorylation of ERK, recruitment of β-arrestin (either or both ofβ-arrestin 1 or β-arrestin 2) to the receptor, inhibition of adenylylcyclase (leading to lowering of intracellular cAMP levels), anddisplacement of probes (e.g., radiolabeled) already bound to thereceptor. Examples of physical effects include conformational changes inthe receptor, changes in the affinity and/or specificity of ligandbinding, effects on receptor dimerization (homodimerizatoin orheterodimerization), effects on receptor trimerization(homotrimerizatoin or heterotrimerization), receptor degradation andreceptor translocation. Examples of chemical effects include effects onavailability of hydrogen-bonding networks in the active site. Samples orassays comprising ORs that are treated with a potential activator,inhibitor, or modulator are compared to control samples without theinhibitor, activator, or modulator to examine the extent of activation,inhibition or modulation.

The effects of the compounds described herein, upon the function of anOR, can be measured by examining any of the parameters described above.Any suitable physiological change that affects OR activity can be usedto assess the influence of a compound on an OR and on natural ornon-natural ligand-mediated OR activity. When the functionalconsequences are determined using intact cells or animals, it is alsopossible to measure a variety of effects such as changes in the levelsof intracellular second messengers such as cAMP.

Modulators of OR activity are tested using OR polypeptides as describedabove (e.g., mu OR, kappa OR, delta OR and ORL1), either recombinant ornaturally occurring. The protein can be isolated, expressed in a cell,expressed in a membrane derived from a cell, expressed in tissue orexpressed in an animal. For example, neuronal cells, cells of the immunesystem, transformed cells, or membranes can be used to test the GPCRpolypeptides described herein. Modulation is tested using an in vitro orin vivo assay, as described herein or known in the art. Signaltransduction can be examined in vitro with soluble or solid statereactions, using a chimeric molecule such as an extracellular domain ofa receptor covalently linked to a heterologous signal transductiondomain, or a heterologous extracellular domain covalently linked to thetransmembrane and or cytoplasmic domain of a receptor. Furthermore,ligand-binding domains of a protein of interest can be used in vitro insoluble or solid state reactions to assay for ligand binding.

Ligand binding to an OR, an OR domain, or a chimeric protein can betested in a number of formats. Binding can be performed in solution, ina bilayer membrane, attached to a solid phase, in a lipid monolayer, orin vesicles. In certain of the assays described herein, the binding ofthe natural ligand to its receptor is measured in the presence of acandidate modulator. Alternatively, the binding of a candidate modulatorcan be measured in the presence of the natural ligand. Competitiveassays that measure the ability of a compound to compete with binding ofthe natural ligand to the receptor can be used. Competitive assays thatmeasure the ability of a compound to compete with the binding of apositive allosteric modulator (PAM) can also be used, e.g., to identifypotential silent allosteric modulators (SAMs). Binding can be tested bymeasuring, e.g., changes in spectroscopic characteristics (e.g.,fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape)changes, or changes in chromatographic or solubility properties.

Modulators can also be identified using assays involving beta-arrestinrecruitment. β-arrestin is a protein that is distributed throughout thecytoplasm in unactivated cells. Ligand binding to an appropriate ORresults in redistribution of β-arrestin from the cytoplasm to the cellsurface, where it associates with the OR. Thus, receptor activation andthe effect of candidate modulators on ligand-induced receptoractivation, can be assessed by monitoring β-arrestin recruitment to thecell surface.

Other assays for MOR activation are known in the art. See, for example,WO 2012/129495.

Compounds

The MOR PAMs and MOR SAMs provided herein are compounds having thestructure:

Wherein

A1 is null, CH₂, CHR₁, CR₂R₃, CH, CR₄, CO, O, S, SO, SO₂, NH or NR₅;

A2 is null, CH₂, CHR₆, CR₇R₈, CH, CR₉, CO, O, S, SO, SO₂, NH or NR₁₀;

A3 is null, CH₂, CHR₁₁, CR₁₂R₁₃, CH, CR₁₄, CO, O, S, SO, SO₂, NH, NR₁₅;

A4 is null, CH₂, CHR₁₆, CR₁₇R₁₈, CH, CR₁₉, CO, O, S, SO, SO₂, NH, NR₂₀;

A5 is null, CH₂, CHR₂₁, CR₂₂R₂₃, CH, CR₂₄, CO, O, SO, SO₂, NH, NR₂₅;

A6 is CH or CR₂₆;

A7 is SO₂R₂₇, SOR₂₈, CHR₂₉, R₃₀, CH₂R₃₁, COR₃₂, CONHR₃₃, CONR₃₄R₃₅,alkyl, branched alkyl, substituted alkyl, aryl, substituted aryl,cyclic, substituted cyclic, heterocyclic, substituted heterocyclic,heteroaryl, substituted heteroaryl or a small substitution group;

A8 is alkly, branched alkyl, substituted alkyl, aryl, substituted aryl,cyclic, substituted cyclic, heterocyclic, substituted heterocyclic,heteroaryl, substituted heteroaryl, biaryl, substituted biaryl,heterobiaryl, substituted heterobiaryl or a small substitution group;and

R1 through R35 are independently alkyl, branched alkyl, halogenatedalkyl, branched halogenated alkyl, branched alkylcarbonyl, halogenatedalkylcarbonyl, branched halogenated alkylcarbonyl, arylcarbonyl alkenyl,substituted alkenyl, alkynyl, ether or a small substitution group;further wherein no more than 4 of A1-A5 are null.

In certain embodiments, one or more of the hydrogen atoms attached toA1, A2, A3, A4, A5 or A6 can be replaced with a deuterium (D) atom.

No more than two heteroatoms (e.g., O, N, S) can be present withinA1-A4; and O—O, S—O, S—S and S—N bonds within A1 through A6 areexcluded.

In certain embodiments, any one or more of A7, A8 or R1-R35 are a smallsubstitution group, selected from cyano, halogen, lower alkyl (e.g.,C1-C3 alkyl), branched lower alkyl (e.g., isopropyl), halogenated alkyl,hydroxyl, oxyalkyl, alkyloxy, amino, alkylamino, dialkylamino,mercaptanyl, alkylmercaptanyl, alkylsulfonyl, aminosulfonyl,alkylaminosulfonyl, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, aryl, arylalkyl, carbocycle,carbocycle-alkyl, F, Cl, Br, CH₃, CH₂CH₃, CH₂F, CHF₂, CF₃, n-Pr, n-Bu,i-Bu, sec-Bu, iPr, t-Bu, CN, OH, OMe, OEt, O-iPr, OCF₂H, OCF₃, NH₂,NHMe, NMe₂, methoxycarbonyl, methanesulfonyl, phenyl, benzyl, MeSO₂,formyl or acetyl.

Any one or more of the bonds between N and A1, A1 and A2, A2 and A3, A3and A4, A4 and A5, A5 and A6, and A6 and N can be a double bond,provided that the distribution of double bonds in the molecule resultsin a stable structure.

In certain embodiments, the compound of Formula I is a six-membered,nitrogen-containing ring (i.e., a piperidine ring) with substituents A7and A8.

In certain embodiments, the compound of Formula I is a four, five, sixor seven-membered ring that is fused to a second ring, forming aspirocycle, a bridged bicycle or a fused bicycle.

In certain of the bicyclic compounds provided herein a spirocyclicbicycle is formed. For spirocyclic bicycles, the point of attachment ofthe second ring can be at any of A1-A5. The second ring can be anycarbocycle, substituted carbocycle, heterocarbocycle or substitutedheterocarbocylcle of ring size 3, 4, 5, or 6. A nonlimiting list ofappropriate carbocyles is provided in Table 2. Exemplary spirocyclicbicycles of Formula 1 are shown below:

In certain of the bicyclic compounds provided herein a bridged bicycleis formed, in which A1 can be connected to any of A4, A5 or A6 by acarbon bridge, or A6 can be connected to A4 or A2 by a carbon bridge.The carbon bridge can be, for example a methylene (—CH₂—), ethylene(—CH₂CH₂—) or propylene (—CH₂CH₂CH₂—) bridge, or a substituted versionthereof. For bridges terminating in either A1 or A6, a heteroatom cannotbe present in the bridge.

In additional bicyclic compounds provided herein, A2 is connected to A5by a carbon, oxygen or nitrogen bridge. The carbon bridge can be, forexample, a methylene, ethylene, or propylene bridge or a substitutedversion thereof. The oxygen bridge can be, for example, an ether (—O—)bridge; and the nitrogen bridge can be, for example, an amine bridge(e.g., —NH— or —NR₃₆—).

In the amine bridge, R36 can be one of cyano, halogen, hydroxyl,alkyloxy, alkyl, branched alkyl, halogenated alkyl, branched halogenatedalkyl, aryl, arylalkyl, carbocycle, carbocycle-alkyl, alkylcarbonyl,branched alkylcarbonyl, halogenated alkylcarbonyl, branched halogenatedalkylcarbonyl, arylcarbonyl alkenyl, substituted alkenyl, alkynyl,alkoxycarbonyl, ether or a small substitution group. The smallsubstitution group is selected from cyano, halogen, alkyl, branchedalkyl, halogenated alkyl, hydroxyl, alkyloxy, amino, alkylamino,dialkylamino, mercaptanyl, alkylmercaptanyl, alkylsulfonyl,aminosulfonyl, alkylaminosulfonyl, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, aryl,arylalkyl, carbocycle, carbocycle-alkyl, F, Cl, Br, CH₃, CH₂CH₃, CH₂F,CHF₂, CF₃, n-Pr, n-Bu, i-Bu, sec-bu, i-Pr, t-Bu, CN, OH, OMe, OEt,O-iPr, OCF2H, OCF3, NH2, NHMe, NMe2, methoxycarbonyl, methanesuflonyl,phenyl, benzyl, MeSO₂, formyl, and acetyl.

Exemplary bridged bicycles of Formula 1 are shown below:

In certain of the bicyclic compounds provided herein a fused bicycle isformed. The point of attachment of the second ring can be at any ofA1-A2, A2-A3, A3-A4, A2-A4 (when A3 is null), or A4-A5. The second ringcan be any carbocycle, substituted carbocycle, heterocarbocycle orsubstituted heterocarbocylcle of ring size 3, 4, 5, or 6. A non-limitinglist of appropriate carbocyles is provided in Table 2. The second ringcan also be any aryl, substituted aryl, heteroaryl, or substitutedheteroaryl. A non-limiting list of examples of the second ring isprovided in Table 1. Exemplary fused bicycles of Formula 1 are shownbelow:

For spirocyclic and fused bicyclic compounds, the second ring can be,for example, aryl, phenyl, cycloalkyl (e.g., cyclohexyl), pyridine,pyrimidine, furan, thiophene or pyridazine.

In the spirocyclic and fused bicyclic compounds disclosed herein, eitheror both of the rings can be substituted with, for example, one or moreof cyano, halogen, alkyl, branched alkyl, halogenated alkyl, hydroxyl,alkyloxy, formyl, acetyl, amino, alkylamino, dialkylamino, mercaptanyl,alkylmercaptanyl, or a small substitution group. The small substitutiongroup is selected from cyano, halogen, alkyl, branched alkyl,halogenated alkyl, hydroxyl, alkyloxy, oxyalkyl, amino, alkylamino,dialkylamino, mercaptanyl, alkylmercaptanyl, alkylsulfonyl,aminosulfonyl, alkylaminosulfonyl, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, aryl,arylalkyl, carbocycle, carbocycle-alkyl, F, Cl, Br, CH₃, CH₂CH₃, CH₂F,CHF₂, CF₃, n-Pr, n-Bu, i-Bu, sec-Bu, iPr, t-Bu, CN, OH, OMe, OEt, O-iPr,OCF₂H, OCF₃, NH₂, NHMe, NMe₂, methoxycarbonyl, methanesulfonyl, phenyl,benzyl, MeSO₂, formyl or acetyl.

In certain embodiments, provided herein are compounds having thestructure

wherein the definitions of A1, A2, A4-A8, R1-R10, R16-R35 and “smallsubstitution group” are as provided for Formula I above.

In the compounds of Formula Ia, any one or more of the bonds between Nand A1, A1 and A2, A2 and A4, A4 and A5, A5 and A6, and A6 and N can bea double bond, provided that the distribution of double bonds in themolecule results in a stable structure.

In certain embodiments, the compound of Formula Ia is a six-membered,nitrogen-containing ring with substituents A7 and A8.

In certain embodiments, the compound of Formula I is a four, five, sixor seven-membered ring that is fused to a second ring, forming aspirocycle, a bridged bicycle or a fused bicycle, as noted above withrespect to Formula 1.

In additional embodiments, provided herein are compounds of Formula Iawherein A8 is an aromatic (aryl) or heteroaromatic (heteroaryl) ring.Exemplary aryl or heteroaryl rings include phenyl, cycloalkyl (e.g.,cyclohexyl, cycloheptyl), pyridine, pyridazine, pyrimidine and pyrazine.

In additional embodiments, provided herein are compounds having thestructure

wherein the definitions of A1, A2, A4, A6, A7, R1-R10, R16-R20, R26-R35and “small substitution group” are as provided for Formula I above, andA8 is an aromatic (aryl) or heteroaromatic (heteroaryl) ring. Exemplaryaryl or heteroaryl rings include phenyl, cycloalkyl (e.g., cyclohexyl,cycloheptyl), pyridine, pyridazine, pyrimidine and pyrazine.

In further embodiments, provided herein are compounds having thestructure

wherein the definitions of A1, A2, A4, A6, A7, R1-R10, R16-R20, R26-R35and “small substitution group” are as provided for Formula I above, andA8 is an aromatic (aryl) or heteroaromatic (heteroaryl) ring. Exemplaryaryl or heteroaryl rings include phenyl, cycloalkyl (e.g., cyclohexyl,cycloheptyl), pyridine, pyridazine, pyrimidine and pyrazine.

In further embodiments, provided herein are compounds of Formula 1awherein the ring containing N, A1, A2, A4 and A6 is part of a bridgedbicyclic ring system. In these bridged bicyclic compounds, A1 can beconnected to any of A4, A5 or A6 by a carbon bridge, or A6 can beconnected to A4 or A2 by a carbon bridge. The carbon bridge can be, forexample a methylene (—CH₂—), ethylene (—CH₂CH₂—) or propylene(—CH₂CH₂CH₂—) bridge, or a substituted version thereof. For bridgesterminating in either A1 or A6, a heteroatom cannot be present in thebridge.

In additional bridged bicyclic compounds of Formula 1a, A2 is connectedto A5 by a carbon, oxygen or nitrogen bridge. The carbon bridge can be,for example, a methylene, ethylene, or propylene bridge or a substitutedversion thereof. The oxygen bridge can be, for example, an ether (—O—)bridge; and the nitrogen bridge can be, for example, an amine bridge(e.g., —NH— or —NR₃₆—).

In the amine bridge, R36 can be one of cyano, halogen, hydroxyl,alkyloxy, alkyl, branched alkyl, halogenated alkyl, branched halogenatedalkyl, aryl, arylalkyl, carbocycle, carbocycle-alkyl, alkylcarbonyl,branched alkylcarbonyl, halogenated alkylcarbonyl, branched halogenatedalkylcarbonyl, arylcarbonyl alkenyl, substituted alkenyl, alkynyl,alkoxycarbonyl, ether or a small substitution group. The smallsubstitution group is selected from cyano, halogen, alkyl, branchedalkyl, halogenated alkyl, hydroxyl, alkyloxy, amino, alkylamino,dialkylamino, mercaptanyl, alkylmercaptanyl, alkylsulfonyl,aminosulfonyl, alkylaminosulfonyl, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, aryl,arylalkyl, carbocycle, carbocycle-alkyl, F, Cl, Br, CH₃, CH₂CH₃, CH₂F,CHF₂, CF₃, n-Pr, n-Bu, i-Bu, sec-bu, i-Pr, t-Bu, CN, OH, OMe, OEt,O-iPr, OCF₂H, OCF₃, NH₂, NHMe, NMe₂, methoxycarbonyl, methanesuflonyl,phenyl, benzyl, MeSO₂, formyl, and acetyl.

Exemplary bridged bicycles of Formula 1a are shown below:

In further embodiments, provided herein are compounds having thestructure

wherein the definitions of A1, A2, A4, A6, R1-R10, R16-R20, R26-R35 and“small substitution group” are as provided for Formula I above, and A8is an aromatic (aryl) or heteroaromatic (heteroaryl) ring. Exemplaryaryl or heteroaryl rings include phenyl, cycloalkyl (e.g., cyclohexyl,cycloheptyl), pyridine, pyridazine, pyrimidine and pyrazine.

In additional embodiments, provided herein are compounds having thestructure

wherein the definitions of A1, A2, A4, A6, R1-R10, R16-R20, R26-R35 and“small substitution group” are as provided for Formula I above, and A8is an aromatic (aryl) or heteroaromatic (heteroaryl) ring. Exemplaryaryl or heteroaryl rings include phenyl, cycloalkyl (e.g., cyclohexyl,cycloheptyl), pyridine, pyridazine, pyrimidine and pyrazine.

In the compounds of Formulas 1d and 1e, R37 can be one of cyano,halogen, hydroxyl, alkyloxy, oxyalkyl, alkyl, branched alkyl,halogenated alkyl, branched halogenated alkyl, aryl, substituted aryl,arylalkyl, carbocycle, carbocycle-alkyl, alkylcarbonyl, branchedalkylcarbonyl, halogenated alkylcarbonyl, branched halogenatedalkylcarbonyl, arylcarbonyl alkenyl, substituted alkenyl, alkynyl,alkoxycarbonyl, ether or a small substitution group. The smallsubstitution group is selected from cyano, halogen, alkyl, branchedalkyl, halogenated alkyl, hydroxyl, alkyloxy, amino, alkylamino,dialkylamino, mercaptanyl, alkylmercaptanyl, alkylsulfonyl,aminosulfonyl, alkylaminosulfonyl, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, aryl,arylalkyl, carbocycle, carbocycle-alkyl, F, Cl, Br, CH₃, CH₂CH₃, CH₂F,CHF₂, CF₃, n-Pr, n-Bu, i-Bu, sec-bu, i-Pr, t-Bu, CN, OH, OMe, OEt,O-iPr, OCF₂H, OCF₃, NH₂, NHMe, NMe₂, methoxycarbonyl, methanesuflonyl,phenyl, benzyl, MeSO₂, formyl, and acetyl.

In further embodiments, provided herein are compounds of Formula 1awherein the ring containing N, A1, A2, A4 and A6 is part of a bridgedbicyclic ring system; A8 is aryl or heteroaryl; and A7 is —SO₂R³⁷.Exemplary aryl or heteroaryl rings include phenyl, cycloalkyl (e.g.,cyclohexyl, cycloheptyl), pyridine, pyridazine, pyrimidine and pyrazine.R37 is the same as defined above for the compounds of Formulas 1d and1e.

In these bridged bicyclic compounds, A1 can be connected to any of A4,A5 or A6 by a carbon bridge, or A6 can be connected to A4 or A2 by acarbon bridge. The carbon bridge can be, for example a methylene(—CH₂—), ethylene (—CH₂CH₂—) or propylene (—CH₂CH₂CH₂—) bridge, or asubstituted version thereof. For bridges terminating in either A1 or A6,a heteroatom cannot be present in the bridge.

In additional bridged bicyclic compounds of Formula 1a, A2 is connectedto A5 by a carbon, oxygen or nitrogen bridge. The carbon bridge can be,for example, a methylene, ethylene, or propylene bridge or a substitutedversion thereof. The oxygen bridge can be, for example, an ether (—O—)bridge; and the nitrogen bridge can be, for example, an amine bridge(e.g., —NH— or —NR₃₆—).

In the amine bridge, R36 can be one of cyano, halogen, hydroxyl,alkyloxy, alkyl, branched alkyl, halogenated alkyl, branched halogenatedalkyl, aryl, arylalkyl, carbocycle, carbocycle-alkyl, alkylcarbonyl,branched alkylcarbonyl, halogenated alkylcarbonyl, branched halogenatedalkylcarbonyl, arylcarbonyl alkenyl, substituted alkenyl, alkynyl,alkoxycarbonyl, ether or a small substitution group. The smallsubstitution group is selected from cyano, halogen, alkyl, branchedalkyl, halogenated alkyl, hydroxyl, alkyloxy, amino, alkylamino,dialkylamino, mercaptanyl, alkylmercaptanyl, alkylsulfonyl,aminosulfonyl, alkylaminosulfonyl, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, aryl,arylalkyl, carbocycle, carbocycle-alkyl, F, Cl, Br, CH₃, CH₂CH₃, CH₂F,CHF₂, CF₃, n-Pr, n-Bu, i-Bu, sec-bu, i-Pr, t-Bu, CN, OH, OMe, OEt,O-iPr, OCF₂H, OCF₃, NH₂, NHMe, NMe₂, methoxycarbonyl, methanesuflonyl,phenyl, benzyl, MeSO₂, formyl, and acetyl.

In these aforementioned bicyclic compounds, either or both of the ringscan be substituted with, for example, one or more of cyano, halogen,alkyl, branched alkyl, halogenated alkyl, hydroxyl, alkyloxy, formyl,acetyl, amino, alkylamino, dialkylamino, mercaptanyl, alkylmercaptanyl,or a small substitution group. The small substitution group is selectedfrom cyano, halogen, alkyl, branched alkyl, halogenated alkyl, hydroxyl,alkyloxy, amino, alkylamino, dialkylamino, mercaptanyl,alkylmercaptanyl, alkylsulfonyl, aminosulfonyl, alkylaminosulfonyl,alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, aryl, arylalkyl, carbocycle, carbocycle-alkyl, F,Cl, Br, CH₃, CH₂CH₃, CH₂F, CHF₂, CF₃, n-Pr, n-Bu, i-Bu, sec-Bu, iPr,t-Bu, CN, OH, OMe, OEt, O-iPr, OCF₂H, OCF₃, NH₂, NHMe, NMe₂,methoxycarbonyl, methanesulfonyl, phenyl, benzyl, MeSO₂, formyl oracetyl.

In further embodiments, provided herein are compounds having thestructure

wherein A9 is CH or N; A10 is C or N and R38-R43 are H, D, Cl, F, Br,CF₃, —OCF₃, CH₃, CH₂CH₃, CH₂F, CHF₂, n-Pr, i-Pr, n-Bu, iso-Bu, sec-Bu,t-Bu, —CN, —OH, —OCH₃, —OCH₂CH₃, O-iPr, OCF₂H, OCHF₂, OCF₃, NH₂, NHCH₃,N(CH₃)₂, methoxycarbonyl, methanesulfonyl MeSO₂, formyl or acetyl; orR43 is null if A10 is N.

In certain embodiments, in the compound of Formula 2 A9 is CH or N; A10is C or N R38 is Br, Cl, CF₃ or —OCF₃, R39 is H, Cl or F; R40 is H or F;R41 is H or D (deuterium); R42 is H or F; R43 is H or F when A10 is C;and R43 is null when A10 is N.

In additional embodiments of the compound of Formula 2, A9 is CH; A10 isN; R38 is Cl; R39 is F; R40 is H; R41 is H; R42 is H and R43 is null.

In additional embodiments of the compound of Formula 2, A9 is N; A10 isC; R38 is CF₃; R39 is H; R40 is H; R41 is H; R42 is F; and R43 is H.

In additional embodiments of the compound of Formula 2, A9 is N; A10 isC; R38 is —OCF₃; R39 is H; R40 is H; R41 is H; R42 is F; and R43 is H.

In additional embodiments of the compound of Formula 2, A9 is N; A10 isC; R38 is CF₃; R39 is H; R40 is H; R41 is H; R42 is H; and R43 is H.

In additional embodiments of the compound of Formula 2, A9 is N; A10 isC; R38 is CF3; R39 is H; R40 is H; R41 is H; R42 is H; and R43 is F.

In additional embodiments of the compound of Formula 2, A9 is CH; A10 isC; R38 is Br; R39 is F; R40 is F; R41 is H; R42 is H; and R43 is H.

In additional embodiments of the compound of Formula 2, A9 is CH; A10 isC; R38 is Br; R39 is F; R40 is F; R41 is D; R42 is H; and R43 is H.

In additional embodiments of the compound of Formula 2, A9 is CH; A10 isC; R38 is CF₃; R39 is H; R40 is F; R41 is D; R42 is H; and R43 is F.

In further embodiments, provided herein are compounds having thestructure

wherein A11 is CH or N; and R44-R49 are H, D, Cl, F, Br, CF₃, —OCF₃,CH₃, CH₂CH₃, CH₂F, CHF₂, n-Pr, i-Pr, n-Bu, iso-Bu, sec-Bu, t-Bu, —CN,—OH, —OCH₃, —OCH₂CH₃, O-iPr, OCF₂H, OCHF₂, OCF₃, NH₂, NHCH₃, N(CH₃)₂,methoxycarbonyl, methanesulfonyl MeSO₂, formyl or acetyl.

In certain embodiments, in the compound of Formula 3, A11 is CH or N;R44 is Cl, Br or CF₃; R45 is H, Cl, F or OCF₃; R46 is H or F; R47 is Hor D (deuterium); R48 is H or F; and R49 is null, ═CH or F.

In additional embodiments of the compound of Formula 3, A11 is CH; R44is Cl; R45 is F; R46 is H; R47 is H; R48 is H; and R49 is null.

In additional embodiments of the compound of Formula 3, A11 is CH; R44is Br; R45 is F; R46 is H; R47 is H; R48 is H; and R49 is null.

In additional embodiments of the compound of Formula 3, A11 is CH; R44is F; R45 is F; R46 is H; R47 is H; R48 is H; and R49 is null.

In additional embodiments of the compound of Formula 3, A11 is N; R44 isCF₃; R45 is H; R46 is H; R47 is H; R48 is H; and R49 is null.

In additional embodiments of the compound of Formula 3, A11 is CH; R44is CF₃; R45 is H; R46 is H; R47 is H; R48 is F; and R49 is null.

In additional embodiments of the compound of Formula 3, A11 is CH; R44is CF₃; R45 is H; R46 is H; R47 is H; R48 is F; and R49 is ═CH.

In additional embodiments of the compound of Formula 3, A11 is CH; R44is CF₃; R45 is H; R46 is H; R47 is H; R48 is F; and R49 is F.

In additional embodiments of the compound of Formula 3, A11 is CH; R44is CF₃; R45 is H; R46 is H; R47 is H; R48 is H; and R49 is null.

In additional embodiments of the compound of Formula 3, A11 is CH; R44is CF₃; R45 is Cl; R46 is H; R47 is H; R48 is H; and R49 is null.

In additional embodiments of the compound of Formula 3, A11 is CH; R44is Br; R45 is —OCF₃; R46 is H; R47 is H; R48 is H; and R49 is null.

In additional embodiments of the compound of Formula 3, A11 is CH; R44is Br; R45 is Cl; R46 is H; R47 is H; R48 is H; and R49 is null.

In additional embodiments of the compound of Formula 3, A11 is CH; R44is CF₃; R45 is H; R46 is F; R47 is D; R48 is H; and R49 is null.

In further embodiments, provided herein are compounds having thestructure

wherein A12 is CH or N; A13 is CH₂, NH or null; and R50-R57 are H, D,Cl, F, Br, CF₃, —OCF₃, CH₃, CH₂CH₃, CH₂F, CHF₂, n-Pr, i-Pr, n-Bu,iso-Bu, sec-Bu, t-Bu, —CN, —OH, —OCH₃, —OCH₂CH₃, O-iPr, OCF₂H, OCHF₂,OCF₃, NH₂, NHCH₃, N(CH₃)₂, methoxycarbonyl, methanesulfonyl MeSO₂,formyl or acetyl. In addition, R50 and R51 together can form a secondring (aryl or heteroaryl) that is fused to the A12-containing ring. Incertain embodiments, in the compound of Formula 4, A12 is C or N; A13 isCH₂ or null; R50 is Cl, Br or CF₃; R51 is H, F or Cl; R52 is H or F; R53is H, F or CH₃; R54 is H or D; R55 is H or F; R56 is H or Cl; and R57 isH or Cl.

In further embodiments, provided herein are compounds having thestructure

wherein A14 is CH or N; and R58-R60 are H, D, Cl, F, Br, CF3, —OCF₃,CH₃, CH₂CH₃, CH₂F, CHF₂, n-Pr, i-Pr, n-Bu, iso-Bu, sec-Bu, t-Bu, —CN,—OH, —OCH₃, —OCH₂CH₃, O-iPr, OCF₂H, OCHF₂, OCF₃, NH₂, NHCH₃, N(CH₃)₂,methoxycarbonyl, methanesulfonyl MeSO₂, formyl or acetyl.

In certain embodiments, in the compound of Formula 5, A14 is CH or N;R58 is Cl, Br or CF₃; R59 is H, F or Cl; and R60 is H, F or CF₃.

In further embodiments, provided herein are compounds having thestructure

wherein A15 is CH or N; and R61-R66 are H, D, Cl, F, Br, CF₃, —OCF₃,CH₃, CH₂CH₃, CH₂F, CHF₂, n-Pr, i-Pr, n-Bu, iso-Bu, sec-Bu, t-Bu, —CN,—OH, —OCH₃, —OCH₂CH₃, O-iPr, OCF₂H, OCHF₂, OCF₃, NH₂, NHCH₃, N(CH₃)₂,methoxycarbonyl, methanesulfonyl MeSO₂, formyl or acetyl.

In certain embodiments, in the compound of Formula 6, A15 is CH or N;R61 is Cl, Br or CF₃; R62 is H, F, Cl or —OCH₃; R63 is H or F; R64 is Hor F; R65 is H or Cl; and R66 is H or Cl.

In further embodiments, provided herein are compounds having thestructure

wherein A16 is CH or N; A17 is CH₂, NH or null; and R67-R69 are H, D,Cl, F, Br, CF₃, —OCF₃, CH₃, CH₂CH₃, CH₂F, CHF₂, n-Pr, i-Pr, n-Bu,iso-Bu, sec-Bu, t-Bu, —CN, —OH, —OCH₃, —OCH₂CH₃, O-iPr, OCF₂H, OCHF₂,OCF₃, NH₂, NHCH₃, N(CH₃)₂, methoxycarbonyl, methanesulfonyl MeSO₂,formyl or acetyl.

In certain embodiments, in the compound of Formula 7, A16 is CH or N;A17 is CH₂ or null; R67 is H, Cl, F, —OCH₃ or —OCF₃; R68 is Cl, Br, CH₃or CF₃; and when A17 is null, R69 is —CH₃.

In further embodiments, provided herein are compounds having thestructure

wherein X is C, O, S, SO, SO₂, N, NH, NCH₃, NAc, NCO(CMe₂OH), NSO₂Me, orNR72; wherein R72 is alkyl, aryl, heteroaryl; R70 is aryl, heteroaryl,substituted aryl and substituted heteroaryl and R71 is aryl, heteroaryl,substituted aryl and substituted heteroaryl.

An aryl group, as present in any of the compounds disclosed herein, iseither a monocyclic aromatic group or a bicyclic aromatic group, and cancontain heteroatoms in the aromatic group (e.g., heteroaryl).Non-limiting structures of exemplary aryl groups are provided in Table1.

TABLE 1 Exemplary aryl groups

A carbocycle, as present in certain of the compounds disclosed herein,is either a monocyclic or a bicyclic non-aromatic ring system. Table 2provides non-limiting structures of some exemplary carbocycles, whereinX1 and X2 are independently O, S, N, NH or NR70. R70 can be hydroxyl,alkyloxy, alkyl, branched alkyl, halogenated alkyl, branched halogenatedalkyl, aryl, arylalkyl, carbocycle, carbocycle-alkyl, alkylcarbonyl,branched alkylcarbonyl, halogenated alkylcarbonyl, branched halogenatedalkylcarbonyl, arylcarbonyl, alkoxycarbonyl or a small substitutiongroup selected from F, Cl, Br, CH₃, CH₂CH₃, CH₂F, CHF₂, CF₃, n-Pr, n-Bu,i-Bu, sec-Bu, i-Pr, t-Bu, CN, OH, OMe, OEt, 0-i-Pr, methoxycarbonyl,phenyl, benzyl, formyl or acetyl, providing the resulting structure isstable. Although a carbocycle can contain one or more double bond(s),the distribution of double bonds in a carbocycle does not constitute anaromatic ring system.

TABLE 2 Exemplary carbocyclic groups

Signal Bias

Activation of the G-protein-coupled MOR results in a number ofdownstream effects, including GDP/GTP exchange, ERK phosphorylation,recruitment of β-arrestins (i.e., β-arrestin 1, β-arrestin 2 andβ-arrestin 3) and inhibition of cAMP production. A number of studieshave shown that signaling events downstream of GPCR activation havesignificant effects on physiologic processes. Kenakin, T. (2015b)British Pharmacological Society 173:4238-4235. For example, animalknockout studies (Soergel et al., supra and references therein) havedemonstrated the benefits of ligands that inhibit adenylyl cyclaseactivity more strongly than they stimulate β-arrestin recruitment. It isbelieved that activation of the β-arrestin pathway not only negativelyimpacts pain amelioration, but is directly contributory togastrointestinal side effects of opiates. Raehal et al., (2005) J.Pharmacol. Exp. Therapeutics 314:1195-1201; Thompson et al. (2015)Molecular Pharmacology 88:335-346; Rivero et al. (2012) MolecularPharmacology, (2012) 82:178-188; Pradham et al. (2012) British Journalof Pharmacology 167(5):960-969. In fact, it has been clinicallydemonstrated that the therapeutic index for an orthosteric partialagonist of the MOR is increased by biasing the signal toward adenylatecyclase inhibition and away from β-arrestin signaling. Thus, oneapproach to improving the therapeutic efficacy of opiates and opioidshas been to modify orthosteric ligands such that they exhibit selectivesignal bias.

Certain of the MOR PAMs disclosed herein also exhibit selective signalbias, in that they have differential effects on downstream processesresulting from MOR signaling. For example, Table 3 shows that certaincompounds have quantitatively different EC₅₀ and maximal response valuesfor β-arrestin recruitment compared to adenylyl cyclase inhibition.Table 4 shows results of similar analyses of additional compounds.

TABLE 3 Compounds showing selective signal bias β-arr. β-arr. cAMP cAMPNo. EC₅₀ (nM) Max Resp. EC₅₀ (nM) Max Resp. 1 572 138 186 63 2 155 72170 85 14 13,212 111 9,608 43 44 5,670 99 3,735 57 100 522 76 >25,000 25207 1,172 116 969 34 224 11,803 305 409 66 Legend: The first columnprovides the compound number (identified elsewhere herein) of thecompound tested. The second column provides EC₅₀ values for β-arrestinrecruitment; the third column provides maximal response values forβ-arrestin recruitment; the fourth column provides EC₅₀ values foradenylyl cyclase inhibition; and the fifth column provides maximalresponse values for adenylyl cyclase inhibition.

TABLE 4 Activity of compounds on beta-arrestin signaling and oninhibition of cAMP formation. Compound β-arr. β-arr cAMP cAMP No. EC₅₀Max. Resp. EC₅₀ Max. Resp. 1 A F A F 2 A F A F 3 A F A F 4 C F 5 C D C D6 B F A F 7 A F A F 8 A F B F 9 A F A F 10 A F A E 11 A F A E 12 C F B E13 C F C F 14 C F C E 15 C E C E 16 C D C D 17 C D C D 18 C F B D 19 G20 G 21 C F C F 22 C F C D 23 C F C D 24 C F C D 25 C F C D 26 C F C D27 C F C E 28 B F B D 29 C F C E 30 G 31 G 32 C F C D 33 C F C D 34 G 35G 36 G 37 C F C D 38 C F C D 39 G 40 C D 41 C F 42 B E C D 43 B F C D 44C F C E 45 G 46 G 47 G 48 C F C E 49 C F C F 50 C F B D 51 C F B F 52 AF A F 53 C F C F 54 C F C E 55 C F B E 56 C F B E 57 C F C E 58 C F C D59 C F C F 60 A F A E 61 C F C E 62 C F C D 63 C F C E 64 B F B D 65 C FC E 66 C F C E 67 C F B E 68 C F B E 69 C F B D 70 C F C E 71 C F C D 72B F A D 73 C F C D 74 C F C E 75 B F A E 76 C F C D 77 C F C D 78 B F AE 79 C F C D 80 C E C D 81 C E C D 82 C D C D 83 C D C D 84 C E C D 85 CD C D 86 C D C D 87 C F C D 88 C E C D 89 B F C D 90 C D C D 91 C F C D92 B F C D 93 B E C D 94 A F A D 95 C F C D 96 C F C D 97 B F B D 98 C EC D 99 A E A D 100 A F C D 101 B F A D 102 C F B E 103 C F B D 104 B F BD 105 B F B D 106 B F A D 107 C F C D 108 C F C E 109 C F A E 110 C F CF 111 C F 112 G 113 C F 114 C D 115 C E 116 C F 117 C F 118 B E C D 119C E C D 120 C F C F 121 A F A E 122 A F A F 123 B F A E 124 A F A E 125A F A F 126 A F A E 127 A F B E 128 A F B E 129 A F B E 130 A F B E 131B F B D 132 C F B D 133 C F C E 134 C F C D 135 B F A D 136 C F C D 137C D C D 138 C D C D 139 C F C E 140 G 141 C D C D 142 C D C D 143 C D CD 144 C E C D 145 C F B E 146 C F C D 147 C F C D 148 B F C E 149 C F CE 150 A F A E 151 C F C D 152 C F B E 153 C F C D 154 A F A E 155 B F BD 156 C F C E 157 C F C E 158 B F A D 159 B F B E 160 B F B F 161 B F BE 162 C F C E 163 B F A E 164 B F A E 165 C F B F 166 C F C E 167 A F BF 168 C F C F 169 A F A F 170 B F A E 171 B F B E 172 B F A E 173 A F AF 174 B F B E 175 C F C E 176 B F B F 177 C F C E 178 A F B D 179 B F AD 180 C F C E 181 C E C D 182 C E C D 183 C E C D 184 C F C D 185 B F CD 186 C E C D 187 C F C E 188 C F B E 189 B F B F 190 B F A F 191 B F BE 192 C F C D 193 B F C F 194 B F B D 195 B F C D 196 A F A F 197 B F BF 198 B F B F 199 A F B E 200 A F A E 201 C F C F 202 C F C E 203 C F AD 204 B F B F 205 B F A F 206 B F B E 207 B F B D 208 C F B F 209 B F BD 210 C F B E 211 C F C E 212 C F C E 213 C F C D 214 C F B E 215 B F CD 216 A F C D 217 B F C D 218 B E C D 219 B F A F 220 C F B D 222 C F BD 223 C F B E 224 C F A F 225 C F C F 226 C F C F 227 C F C F 228 C F CF 229 C F C F 230 C F C F 231 C E C E 232 C F C F 233 C F C F 234 C F CF 235 C F C F 236 C F C F 221 C D C D 237 C E C D 238 C E C D 239 C E CD 240 C D C E 241 C F C E 242 C D C D 243 C F C E 244 A F A D 245 C D CD 246 B F B D 247 C F C E 248 C F C E 249 C F C E 250 C F C D 251 C F CD 252 C F C E 253 C F C E 254 C D C E 255 C D C F 256 C D C D 257 C F CD 258 C E C F 259 C D C D 260 C D C D 261 B F C D 262 C E C D 263 B F BD 264 C E B F 265 C F C E 266 C E C D 267 C D C E 268 C D C F 269 C F CD 270 C D C D 271 C F B E 272 C D C F 273 C D C D 274 C F B D 275 C D CD 276 C E B D 277 C E C D 278 C F B F 279 C D C F 280 C F C E 281 B F AD 282 B F B D 283 C D C F 284 C D C E 285 B F C E 286 C D C D 287 B F AD 288 C F C E 289 B F C D 290 A F A D 291 C D C F 292 C D C E 293 C F BF 294 C D C F 295 C D C D 296 C D C D 297 C E C E 298 C F A F 299 B F AF 300 C E C F 301 B F A E 302 C E C D 303 C D C E 304 C D C D 305 B F BF 306 C D C F 307 C D C F 308 C E C F 309 C E C D 310 C F B F 311 C F BF 312 A F A F 313 C F B E 326 A D A D 327 A D A D 328 A D A D 329 A D AD 330 A D A D 331 A D A D 332 A D A D 333 A D A D 334 A D A D 335 A D AD 336 A D A D 337 A D A D 338 A D A D 339 A D A D 340 A D A D 341 A D AD 343 A D A D 344 A D A D 345 A D A D 346 A D A D 347 A D A D 348 A D AD 349 A D A D 350 A D A D 351 A D A D 352 A D A D 353 A D A D 354 A D AD 355 A D A D 356 A D A D 357 A D A D 358 A D A D 359 A D A D 360 A D AD 361 A D A D 362 A D A D 363 A D A D 364 A D A D 365 A D A D 366 A D AD 368 A D A D 369 A D A D 370 A D A D 371 A D A D 372 A D A D 373 A D AD 374 A D A D 375 A D A D 376 A D A D 377 A D A D 378 A D A D 379 A D AD 380 A D A D 381 A D A D 382 A D A D 388 A D A D 389 A D A D 390 A D AD 391 A D A D 392 A D A D 393 A D A D 395 A D A D 396 A D A D 400 A D AD 401 A D A D 402 A D A D 403 A D A D 404 A D A D 405 A D A D 406 A D AD 407 A D A D 408 A D A D 409 A D A D 410 A D A D 411 A D A D 412 A D AD 413 A D A D 414 A D A D 415 A D A D 417 A D A D 420 A D A D 421 A D AD 422 A D A D 423 A D A D 424 C F C D 425 C F B E 443 C F C D 444 C F CD 445 C E C D 446 C F C E 447 C D C D 448 C F C D 449 C F C D 450 C F CE 451 C F C D 452 C E C D 453 C D C D 454 C E C D 455 C E C D 456 C F CE 457 C F C E 458 C E C D 459 C F C D 460 C F C F 461 C F C E 462 C E CD 463 C D C D 464 C E C E 465 C E C D 466 C F C D 467 C F C D 468 C F CF 469 C F C D 470 C F C E 471 C F C D 473 C E C D 474 C D C D 475 C F CD 476 C E C D 477 C F C D 478 C F C D 479 C F C D 480 C D C D 481 C D CD 482 C D C D 483 C D C D Legend: The first column provides the compoundnumber (identified elsewhere herein) of the compound tested. The secondcolumn provides EC₅₀ values for β-arrestin recruitment; the third columnprovides maximal response values for β-arrestin recruitment; the fourthcolumn provides EC₅₀ values for adenylyl cyclase inhibition; and thefifth column provides maximal response values for adenylyl cyclaseinhibition. EC₅₀ values are coded as follows: A represents an EC₅₀ < 700nM; B represents an EC₅₀ between 700 nM and 2.1 μM; and C represents anEC 50 > 2.1 μM. Maximal response values are coded as follows: Drepresents a maximal response of < 40%; E represents a maximal responseof 40-60%; and F represents a maximal response of > 60%. G represents <50% inhibition of β-arrestin recruitment at a static concentration of 5μM, for compounds that were not tested for adenylyl cyclase inhibition..

Thus, in addition to previous approaches in which an orthosteric ligandis modified to endow it with selective signal bias, the compositions andmethods described herein provide an improved alternative which combinesthe benefits of allosteric modulation with the advantage of selectivesignal bias by the allosteric modulator.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thecompositions and compounds described herein, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In addition, the materials, methods, andexamples are illustrative only not intended to be limiting. Otherfeatures and advantages of the compositions and compounds describedherein will be apparent from the detailed description, reaction schemes,examples and claims.

REACTION SCHEMES

The following representative schemes illustrate how compounds describedherein can be prepared. The specific solvents and reaction conditionsreferred to are illustrative and are not intended to be limiting.Compounds not described are either commercially available or are readilyprepared by one skilled in the art using available starting materials.

Method 1

Step 1: Magnesium powder (1.2 eq) was added to a round-bottom flask andflame-dried under vacuum. Once cooled down to room temperature, thevessel was back-filled with nitrogen, a crystal of iodine was added andthe flask was flame-dried again under vacuum to sublime the iodine. Oncethe flask has cooled down to room temperature, THF (1.0 M vs. aryliodide) was added followed by the aryl iodide (1.0 eq). The resultingsuspension was then refluxed for 30 min before being cooled down to roomtemperature and then 0° C.

A separate flask was flame-dried under vacuum and back-filled withnitrogen before being charged with CuI (1.1 eq) and 4-chlorobutyrylchloride (n=1, 1.1 eq) or 5-chlorovaleryl chloride (n=2, 1.1 eq) or6-chlorocaproyl chloride (n=3, 1.1 eq) in THF (0.5 M vs. aryl iodide)and cooled down to 0° C. To this solution was slowly transferred viacannula under nitrogen the freshly prepared Grignard reagent. Theresulting mixture was allowed to warm up to room temperature and stirreduntil completion (approximately 2 hours) as determined by mass spectralanalysis. Upon completion, the reaction mixture was quenched withsaturated aqueous NH₄Cl and extracted with diethyl ether (3 times). Thecombined organic layers were washed with saturated aqueous NaCl, driedover Na₂SO₄, filtered and the volatiles were evaporated under reducedpressure. The crude residue was purified by flash chromatography onsilica.

Step 2. To an oven-dried vial equipped with a Teflon septum and magneticstir bar was added the required alkyl chloride (1.0 eq) inN,N-dimethylacetamide (0.4 M vs. alkyl chloride) and NaN₃ (2.0 eq). Thevial was sealed and the reaction mixture was heated to 60° C. for 2hours. Upon completion of the reaction, the solution was cooled down toroom temperature, diluted with saturated aqueous NaCl and extracted withEt₂O (3 times). The combined organic layers were washed with sat. aq.NaCl, dried over Na₂SO₄, filtered and the volatiles were evaporatedunder reduced pressure. The crude residue was purified by flashchromatography on silica.

Step 3. An oven-dried vial equipped with a Teflon septum and magneticstir bar was charged with the appropriate azide (1.0 eq) in THF (0.2 Mvs. azide). Triphenylphosphine (2.0 eq) was then added and the reactionmixture was stirred for 2 hours. Water was added to the reaction mixture(1.0 M vs. azide) and the reaction mixture was stirred for 16 hours. Thereaction mixture was evaporated to dryness and the crude residue wasdissolved in Et₂O before being cooled down to 0° C. The precipitate wasfiltered out and this process was repeated two more times before thefiltrate was concentrated under reduced pressure. The crude residue wasused in the next step without further purification.

Step 4: The crude amino ketone was taken up in MeOH (0.2 M vs. ketone)and cooled down to −20° C. before adding NaBH₄ (10.0 eq) in one portion.The resulting suspension was allowed to warm to room temperature andstirred until completion before being quenched with sat. aq. NaHCO₃. Thereaction mixture was extracted with DCM (3 times). The combined organiclayers were washed with sat. aq. NaCl, dried over Na₂SO₄, filtered andthe volatiles were evaporated under reduced pressure. The crude residuewas dissolved in Et₂O (0.1 M vs. amine) and HCl was added (10.0 eq, 2.0N in Et₂O) to precipitate the desired material as the hydrochloridesalt.

Step 5a: Synthesis of sulfonamides. An oven-dried vial equipped with aTeflon septum and magnetic stir bar was charged with the aminehydrochloride salt (1.0 eq), DCE (0.1 M vs. amine) andN,N-diisopropylethylamine (3.0 eq). The reaction mixture was cooled downto 0° C. before the appropriate sulfonyl chloride (1.2 eq) was added.The reaction mixture was stirred while warming up to room temperature.Upon completion of the reaction, the reaction mixture was diluted with0.1 N aq. HCl and extracted with DCM (3 times). The combined organiclayers were washed with sat. aq. NaCl, dried over Na₂SO₄, filtered andthe solvents were evaporated under reduced pressure. The crude residuewas purified by preparative LC-MS.

Step 5b: Synthesis of heteroanilines: An oven-dried vial equipped with aTeflon septum and magnetic stir bar was charged with the aminehydrochloride salt (1.0 eq), 1-butanol (0.1 M vs. amine),N,N-diisopropylethylamine (3.0 eq) and the appropriatechloro-heteroarene (1.2 eq). The reaction mixture was stirred at 50° C.for 14 h. Upon completion of the reaction, solvents were evaporatedunder reduced pressure. The crude residue was purified by preparativeLC-MS.

Step 5c: Synthesis of benzvlic amines: An oven-dried vial equipped witha Teflon septum and magnetic stir bar was charged with the aminehydrochloride salt (1.0 eq), MeCN (0.1 M vs. amine), Cs₂CO₃ (3.0 eq) andKI (0.1 eq). The appropriate benzyl bromide (1.0 eq) was added dropwise(neat or as 1 M solution in MeCN). The reaction mixture was stirred at80° C. Upon completion of the reaction, the reaction mixture was cooleddown to room temperature, diluted with sat. aq. Na₂CO₃ and extractedwith EtOAc (3 times). The combined organic layers were washed with sat.aq. NaCl, dried over Na₂SO₄, filtered and the solvents were evaporatedunder reduced pressure. The crude residue was purified by preparativeLC-MS.

Step 5d: Synthesis of poorly reactive heteroanilines: An oven-dried vialequipped with a Teflon septum and magnetic stir bar was charged with theamine hydrochloride salt (1.0 eq), anhydrous NMP (0.1 M vs. amine),N,N-diisopropylethylamine (3.0 eq), the appropriate chloro-heteroarene(5.0 eq) and potassium fluoride on alumina (5.0 eq, 40% w/w loading).The reaction mixture was stirred at 180° C. for 18 h. Upon completion ofthe reaction, solids were filtered off and the solvents were evaporatedunder reduced pressure. The crude residue was purified by preparativeLC-MS.

Method 2

Step 1. Int-A can also be intercepted via the following route, withfinal compounds being attained by following the Method A1 Steps 2-5.

A round-bottom flask was flame-dried under vacuum and backfilled withnitrogen before being charged with the required arene (1.0 eq) in DCM (1M vs. ketone). Aluminum trichloride (1.1 eq) was added in one portionfollowed by the dropwise addition of 4-chlorobutyryl chloride (n=1, 1.1eq) or 5-chlorovaleryl chloride (n=2, 1.1 eq) or 6-chlorocaproylchloride (n=3, 1.1 eq). The reaction mixture was heated to reflux for 2hours. Upon completion of the reaction, the reaction mixture was pouredonto cold 3N aq. HCl (2x V_(DCM)) and extracted with DCM (3 times). Thecombined organic layers were washed with sat. aq. NaHCO₃ (1 time) andsat. aq. NaCl (1 time), dried over Na₂SO₄, filtered and the solventswere evaporated under reduced pressure. The crude residue was purifiedby flash chromatography on silica.

The final product was obtained following steps 2, 3, 4, 5a, 5b, 5c or 5dfrom Method 1.

Method 3

Int-C can also be intercepted via the following route, with finalcompounds being attained by following Method 1, steps 5a-d.

Step 1: An oven-dried vial equipped with a Teflon septum and magneticstir bar was charged with Ir[dF(CF₃)ppy]₂(dtbbpy)PF₆ (0.01 eq),NiCl₂.glyme (0.1 eq), 4,4′-di-tert-butyl-2,2′-bipyridyl (0.15 eq), thedesired aryl halide (1.0 eq), the appropriate Boc-protected amino acid(1.5 eq), Cs₂CO₃ (1.5 eq) and DMF (0.02 M vs. aryl bromide). Thereaction mixture was degassed by bubbling with nitrogen for 20 min, thenirradiated with two 26 W fluorescent lamps (at approximately 2 cm awayfrom the light source). After 72 h, the reaction mixture was dilutedwith sat. aq. NaHCO₃ and extracted with Et₂O (3 times). The combinedorganic layers were washed with water (1 time) and sat. aq. NaCl (1time), dried over Na₂SO₄ and concentrated under reduced pressure. Thecrude residue was purified by flash chromatography on silica.

Step 2: An oven-dried vial equipped with a Teflon septum and magneticstir bar was charged with the Boc-protected amine (1.0 eq) and dioxane(0.1 M vs. carbamate). 4.0 N HCl in dioxane (excess) was added and thereaction mixture was stirred until complete deprotection was achieved.Upon completion of the reaction, the reaction mixture was diluted withsat. aq. Na₂CO₃ and extracted with EtOAc (3 times). The combined organiclayers were washed with sat. aq. NaCl, dried over Na₂SO₄, filtered andthe solvents were evaporated under reduced pressure. The crude residuewas dissolved in Et₂O (0.1 M vs. amine) and HCl was added (10.0 eq, 2.0N in Et₂O) to precipitate the desired material as the hydrochloridesalt.

Method 4

Int-B can be attained from either Method 1 or Method 2, with finalcompounds deriving from Int-C2 being attained by following theprocedures detailed in Step 5a, 5b (80° C. instead of 50° C.), 5c or 5dfrom Method 1.

Step 1: A round-bottom flask was flame-dried under vacuum and backfilledwith nitrogen before being charged with the required azido aryl ketone(1.0 eq) in DCM (0.2 M vs. ketone). Triethylamine (2.0 eq) andtriethylsilyltrifluoromethanesulfonate (2.0 eq) were sequentially added.The reaction mixture was stirred until completion (approximately 2hours) before being quenched with sat. aq. NaHCO₃. The reaction mixturewas extracted with DCM and the combined organic layers were washed withsat. aq. NaCl, dried over Na₂SO₄ and quickly pushed through a short plugof silica. Volatiles were then evaporated under reduced pressure. Thecrude residue was then dissolved in MeCN (0.2 M vs. ketone),Selectfluor® was added (1.0 eq) and the resulting solution was stirredat room temperature. Upon completion of the reaction, water was addedand the reaction mixture was extracted with EtOAc (3 times). Thecombined organic layers were washed with sat. aq. NaCl, dried overNa₂SO₄, filtered and the volatiles were evaporated under reducedpressure. The crude residue was purified by flash chromatography.

Step 2: An oven-dried vial equipped with a Teflon septum and magneticstir bar was charged with the appropriate azide (1.0 eq) in THF (0.2 Mvs. azide). Triphenylphosphine (2.0 eq) was then added and the reactionmixture was stirred for 2 hours. Water was added to the reaction mixture(1.0 M vs. azide) and the reaction mixture was stirred for 16 hours. Thereaction mixture was evaporated to dryness and the crude residue wasdissolved in Et₂O before being cooled down to 0° C. The precipitate wasfiltered out and this process was repeated two more times before thefiltrate was concentrated under reduced pressure. The crude residue wasused in the next step without further purification.

Step 3: An oven-dried vial equipped with a Teflon septum and magneticstir bar was charged with the amino ketone (1.0 eq) and DCE (0.1 M vs.amine). The reaction mixture was stirred for 1 hour at room temperaturebefore STAB (2.0 eq) was added in one portion. The reaction mixture wasstirred for 16 hours at room temperature. Upon completion of thereaction, the reaction mixture was diluted with sat. aq. Na₂CO₃ andextracted with EtOAc (3 times). The combined organic layers were washedwith sat. aq. NaCl, dried over Na₂SO₄, filtered and the volatiles wereevaporated under reduced pressure. The crude amine was purified bypreparative LC-MS or salted out as the hydrochloride (see Method 1, Step4).

Method 5

Int-B can be attained from either Method 1 or Method 2. Int-C3 can beattained from Int-B3 by following steps 2 and 3 of Method 4. Finalcompounds deriving from Int-C3 are attained by following the proceduresdetailed in steps 5a, 5b (80° C. instead of 50° C.), 5c or 5d of Method1.

A round-bottom flask was flame-dried under vacuum and backfilled withnitrogen before being charged with the required azido aryl ketone (1.0eq) in DCM (0.2 M vs. ketone). Triethylamine (2.0 eq) andtriethylsilyltrifluoromethanesulfonate (2.0 eq) were sequentially added.The reaction mixture was stirred until completion (approximatively 2hours) before being quenched with sat. aq. NaHCO₃. The reaction mixturewas extracted with DCM and the combined organic layers were washed withsat. aq. NaCl, dried over Na₂SO₄ and quickly pushed through a short plugof silica. Volatiles were then evaporated under reduced pressure. Anoven-dried vial was charged with Togni's reagent(3,3-Dimethyl-1-(trifluoromethyl)-1,2-benziodoxole, 1.5 eq), and CuSCN(0.1 eq) under a nitrogen atmosphere. To these solids were added asolution of the silyl enol ether (1.0 eq) in DMA (0.1 M vs. silyl enolether). The vial was sealed and stirred at 80° C. for 12 hours. Uponcompletion of the reaction, sat. aq. NaCl was added and the reactionmixture was extracted with Et₂O (3 times). The combined organic layerswere washed with sat. aq. NaCl, dried over Na₂SO₄, filtered and thevolatiles were evaporated under reduced pressure. The crude residue waspurified by flash chromatography.

Method 6

Step 1: A round-bottom flask was flame-dried under vacuum and backfilledwith nitrogen before being charged with the required halo arene (1.3 eq)in THF (2 M vs. arene). The reaction mixture was cooled down to −15° C.(ice-acetone bath) before i-PrMgCl (1.2 eq, 2.0 M in THF) was addeddropwise over 10 minutes. The reaction mixture was stirred for 3 hourswhile slowly warming up to 0° C. In a separate round-bottom flask,flame-dried under vacuum and backfilled with nitrogen, was addedN-Boc-2-piperidone (1.0 eq) and THF (0.4 M vs. piperidone) and thereaction mixture was cooled down to −78° C. (dry ice-acetone bath). TheGrignard reagent was added dropwise via cannula to the piperidone over˜20 minutes. Once the transfer was complete, the reaction mixture wasstirred for an additional hour at −78° C. before 4.0 N HCl in dioxane(100.0 eq) was added. The cooling bath was removed and the reactionmixture was stirred for 18 h. Upon completion of the reaction, volatileswere evaporated to dryness under reduced pressure. The crude residue wasdiluted with sat. aq. Na₂CO₃ and extracted with EtOAc (3 times). Thecombined organic layers were washed with sat. aq. NaCl, dried overNa₂SO₄, filtered and the volatiles were evaporated under reducedpressure. The crude residue was used without further purification.

Step 2. An oven-dried vial equipped with a Teflon septum and magneticstir bar was charged with the appropriate amino ketone (1.0 eq) and a1:1 MeOH:H₂O mixture (0.1 M vs. ketone). Selectfluor® (4.0 eq) was addedand the reaction mixture was heated to 80° C. for 16 hours. Uponcompletion of the reaction, the reaction mixture was cooled down to roomtemperature and the volatiles were evaporated to dryness under reducedpressure. The crude residue was diluted with sat. aq. NaCl and extractedwith Et₂O (3 times). The combined organic layers were washed with sat.aq. NaCl, dried over Na₂SO₄, filtered and the volatiles were evaporatedunder reduced pressure. The crude residue was used without furtherpurification.

Step 3. An oven-dried vial equipped with a Teflon septum and magneticstir bar was charged with the amino ketone (1.0 eq) and DCE (0.1 M vs.amine) before NaBH₃CN (1.5 eq) was added in one portion. The reactionmixture was stirred for 3 hours at room temperature. Upon completion ofthe reaction, the reaction mixture was diluted with sat. aq. Na₂CO₃ andextracted with EtOAc (3 times). The combined organic layers were washedwith sat. aq. NaCl, dried over Na₂SO₄, filtered and the volatiles wereevaporated under reduced pressure. The crude amine was purified bypreparative LC-MS or salted out as the hydrochloride (see Method 1, Step4).

Final compounds are obtained by following the procedures detailed insteps 5a, 5b (80° C. instead of 50° C.), 5c or 5d from Method 1.

Method 7

Step 1: A round-bottom flask was flame-dried under vacuum and backfilledwith nitrogen before being charged with 2-piperidone (1.1 eq) in THF(0.5 M vs. piperidone). The reaction mixture was cooled down to −78° C.(dry ice-acetone bath) before n-BuLi (2.2 eq, 1.0 M in THF) was addeddropwise over 15 minutes. The reaction mixture was stirred for 15minutes at −78° C. then 45 minutes at 0° C. The reaction mixture wascooled down to −78° C. the desired alkyl halide (1.0 eq) was addeddropwise as a 1.0 M solution in THF. The reaction mixture was stirred at−78° C. for 15 minutes (iodide) or 1 hour (bromide) beforedi-tert-butyl-dicarbonate (1.35 eq) was added dropwise as a 1.0 Msolution in THF. The reaction mixture was stirred for 15 minutes at −78°C. before being quenched with sat. aq. NH4Cl and the aqueous layer wasextracted with Et₂O (3 times). The combined organic layers were washedwith sat. aq. NaCl, dried over Na₂SO₄, filtered and the volatiles wereevaporated under reduced pressure. The crude residue was purified byflash chromatography on silica gel.

Step 2: A round-bottom flask was flame-dried under vacuum and backfilledwith nitrogen before being charged with the required halo arene (1.3 eq)in THF (2.0 M vs. arene). The reaction mixture was cooled down to −15°C. (ice-acetone bath) before i-PrMgCl (1.2 eq, 2.0 M in THF) was addeddropwise over 10 minutes. The reaction mixture was stirred for 3 hourswhile slowly warming up to 0° C. In a separate round-bottom flask,flame-dried under vacuum and backfilled with nitrogen, was added theappropriate N-Boc-3-alkyl-2-piperidone (1.0 eq) and THF (0.4 M vs.piperidone) and the reaction mixture was cooled down to −78° C. (dryice-acetone bath). The Grignard reagent was added dropwise via cannulato the piperidone over ˜20 minutes. Once the transfer was complete, thereaction mixture was stirred for an additional 2 hours at −78° C. beforeTFA (5.0 eq) was added. The reaction mixture was removed from thecooling bath and stirred for 1 h. Upon reaching room temperature,volatiles were evaporated to dryness under reduced pressure. The cruderesidue was taken up in DCM (0.5 M vs ketone) and cooled down to 0° C.before TFA (half the volume of DCM) was added dropwise. The reactionmixture was stirred at room temperature until complete deprotection hasoccurred. Upon completion of the reaction, the volatiles were evaporatedto dryness under reduced pressure. The crude residue was diluted withsat. aq. Na₂CO₃ and extracted with Et₂O (3 times). The combined organiclayers were washed with sat. aq. NaCl, dried over Na₂SO₄, filtered andthe volatiles were evaporated under reduced pressure. The crude residuewas used without further purification.

Step 3: An oven-dried vial equipped with a Teflon septum and magneticstir bar was charged with the amino ketone (1.0 eq) and DCE (0.1 M vs.amine). The reaction mixture was stirred for 1 hour at room temperaturebefore STAB (2.0 eq) was added in one portion. The reaction mixture wasstirred for 16 hours at room temperature. Upon completion of thereaction, the reaction mixture was diluted with sat. aq. Na₂CO₃ andextracted with EtOAc (3 times). The combined organic layers were washedwith sat. aq. NaCl, dried over Na₂SO₄, filtered and the volatiles wereevaporated under reduced pressure. The crude amine was purified bypreparative LC-MS or salted out as the hydrochloride (see method 1, Step4).

The final target was obtained following step 5a, 5b, 5c or 5d fromMethod 1.

Method 8

Step 1: A round-bottom flask was flame-dried under vacuum and backfilledwith nitrogen before being charged with the required arene (1.0 eq) in1,2-DCE (0.5 M vs. arene). Ethyl 5-chloro-5-oxopentanoate (1.1 eq) andanhydrous AlCl₃ (2.0 eq) were subsequently added to the reactionmixture. The resulting suspension was stirred at 70° C. for 2 hours.Upon completion of the reaction, as judged by TLC, the reaction mixturewas cooled down to room temperature and poured onto ice. The layers wereseparated and the aqueous layer was extracted with DCM (2 times). Thecombined organic layers were washed with iN aq. HCl (2 times), water andsat. aq. NaCl, dried over Na₂SO₄, filtered and the volatiles wereevaporated under reduced pressure. The crude residue was used withoutfurther purification.

Step 2. A round-bottom flask was flame-dried under vacuum and backfilledwith nitrogen before being charged with LiAlH₄ (x g, 1.5 eq) in Et₂O(0.4 M vs. A1). The previously obtained keto-ester (1.0 eq) in Et₂O (0.5M vs. ketone) was added dropwise over 30 minutes. Upon completion of theaddition, the reaction mixture was refluxed and stirred for 5 h. Aftercompletion of the reaction, as judged by TLC, the reaction mixture wascooled down to 0° C. and worked-up following the Fieser process, slowlyadding x mL of H₂O, x mL of 15% aq. NaOH and 2x mL of H₂O, followed bydrying with MgSO₄, stirring for 1 hour, filtration and evaporation ofthe volatiles. The crude residue was purified by silica gel flash columnchromatography.

Step 3. A solution of the diol (1.0 eq) in DCM (0.3 M) was slowly addedto a suspension of PCC (4.0 eq) in DCM (0.7 M) over 20 minutes. Afterstirring for 2 hours, Celite® was added followed by Et₂O (2x V_(DCM))and hexanes (1x V_(DCM)). The reaction mixture was stirred for 20minutes before bring filtered through a plug of Celite® to remove mostof the chromium-containing salts. The volatiles were evaporated and thecrude residue was purified by silica gel column flash chromatography toprovide the desired ketoaldehyde.

Step 4: A round-bottom flask containing the previously synthesizedketoaldehyde (1.0 eq), aniline (1.0 eq) and acetic acid (1.2 eq) in MeOH(0.25 M) was stirred for 30 minutes at room temperature. NaBH₃CN (2.0eq) was added and the reaction mixture was heated up to 40° C. for 4hours. Upon completion of the reaction, as judged by TLC, the reactionmixture was quenched with AcOH and evaporated to dryness. The cruderesidue was purified by preparative LC-MS to afford the desiredpiperidine product.

Separation of Enantiomers

Racemic int-C variants were provided to Lotus Separations LLC ofPrinceton, N.J. for chiral resolution. In general, super critical fluidchromatography was performed using an appropriate column (such as LuxCellulose-4 (2×25 cm)). An examplary eluent used is 10% isopropanol/CO₂,100 bar pumping at 70 mL/min and monitored at 220 nm.

Example Compounds

Example compound 1 was synthesized according to Method 1 using2-chloro-1-fluoro-4-iodobenzene as the aryl iodide and 5-chlorovaleroylchloride in Step 1; and 2-(bromomethyl)-6-chloropyridine as the benzylbromide in Step 5c. ¹H NMR (500 MHz, DMSO-d6), δ (ppm): 7.82 (t, 1H,J=7.8 Hz), 7.58 (dd, 1H, J=7.4, 2.1 Hz), 7.41 (m, 2H), 7.34 (m, 2H),3.55 (d, 1H, J=15.0 Hz), 3.28 (dd, 1H, J=11.0, 2.8 Hz), 3.07 (d, 1H,J=15.0 Hz), 2.85 (dd, 1H, J=11.7, 3.6 Hz), 2.10 (td, 1H, J=11.9, 2.8Hz), 1.71 (ddt, 2H, J=15.3, 12.8, 3.1 Hz), 1.52 (m, 3H), 1.33 (qt, 1H,J=13.5, 3.9 Hz). ¹³C NMR (125 MHz, DMSO-d6), δ (ppm): 160.5, 157.1,155.1, 149.2, 142.7 (d, J=4 Hz), 140.1, 129.2, 127.7 (d, J=7 Hz), 122.3,121.2, 119.3 (d, J=18 Hz), 116.9 (d, J=21 Hz), 66.2, 60.0, 53.3, 36.3,25.4, 24.4.

Example compound 2 was synthesized according to Method 3 using2-bromo-6-(trifluoromethyl)pyridine as the aryl bromide andN-Boc-pipecolic acid as the Boc-protected amino-acid.2-(Bromomethyl)-4-chloro-1-fluorobenzene was used as the benzyl bromidein Step 5c. ¹H NMR (500 MHz, DMSO-d6), δ (ppm): 8.06 (t, 1H, J=7.8 Hz),7.84 (d, 1H, J=7.9 Hz), 7.77 (d, 1H, J=7.5 Hz), 7.40 (dd, 1H, J=6.3, 2.8Hz), 7.32 (ddd, 1H, J=8.8, 4.4, 2.8 Hz), 7.15 (t, 1H, J=9.2 Hz), 3.44(dd, 1H, J=11.1, 2.9 Hz), 3.37 (d, 1H, J=14.2 Hz), 3.17 (d, 1H, J=14.1Hz), 2.88 (dt, 1H, J=11.5, 3.5 Hz), 2.12 (td, 1H, J=11.8, 2.8 Hz), 1.75(m, 2H), 1.64 (dq, 1H, J=13.0, 2.9 Hz), 1.53 (m, 2H), 1.36 (m, 1H). ¹³CNMR (125 MHz, DMSO-d6), δ (ppm): 165.2, 160.7, 158.8, 146.1 (q, J=34Hz), 139.6, 130.8 (d, J=5 Hz), 129.1 (d, J=9 Hz), 128.5 (d, J=3 Hz),127.8 (d, J=16 Hz), 125.6, 122.1 (q, J=274 Hz), 119.8 (d, J=3 Hz), 117.5(d, J=24 Hz), 69.3, 52.9, 52.2, 34.9, 25.6, 24.3.

Example compound 3 was synthesized according to Method 3 using2-bromo-6-(trifluoromethyl)pyridine as the aryl bromide andN-Boc-pipecolic acid as the Boc-protected amino-acid.4-Chlorophenylsulfonyl chloride was used as the sulfonyl chloride inStep 5a. ¹H NMR (500 MHz, DMSO-d6), δ (ppm): 8.08 (t, 1H, J=7.8 Hz),7.74 (dd, 2H, J=7.9, 3.6 Hz), 7.65 (m, 2H), 7.52 (m, 2H), 5.29 (dd, 1H,J=6.1, 2.1 Hz), 3.80 (ddd, 1H, J=13.4, 4.4, 2.5 Hz), 3.39 (td, 1H,J=12.7, 3.3 Hz), 2.14 (ddt, 1H, J=12.6, 4.1, 2.5 Hz), 1.68 (tdd, 1H,J=13.3, 5.8, 3.8 Hz), 1.59 (m, 1H), 1.44 (m, 1H), 1.28 (m, 2H). ¹³C NMR(125 MHz, DMSO-d6), δ (ppm): 160.8, 145.8 (q, J=34 Hz), 139.8, 139.0,138.0, 129.7 (2x C), 128.9 (2x C), 126.1, 122.9 (q, J=273 Hz), 119.5 (d,J=3 Hz), 56.3, 43.1, 28.9, 24.5, 18.3.

Example compound 4 was synthesized according to Method 2 using1-bromo-2-methoxybenzene as the arene and 5-chlorovaleroyl chloride inStep 1. 4-Chlorophenylsulfonyl chloride was used as the sulfonylchloride in Step 5a. ¹H NMR (500 MHz, DMSO-d6), δ (ppm): 7.88 (m, 2H),7.68 (m, 2H), 7.39 (dd, 1H, J=2.3, 0.9 Hz), 7.29 (ddd, 1H, J=8.6, 2.4,1.0 Hz), 7.10 (d, 1H, J=8.6 Hz), 5.12 (t, 1H, J=3.6 Hz), 3.84 (s, 3H),3.75 (m, 1H), 2.92 (ddd, 1H, J=14.2, 12.7, 3.0 Hz), 2.14 (dd, 1H,J=14.4, 3.5 Hz), 1.41 (m, 3H), 1.22 (tdd, 1H, J=14.2, 10.6, 5.7 Hz),1.08 (tdt, 1H, J=12.3, 8.2, 4.4 Hz). ¹³C NMR (125 MHz, DMSO-d6), δ(ppm): 154.7, 140.2, 138.1, 132.9, 131.6, 130.1 (2x C), 129.0 (2x C),127.8, 113.1, 111.3, 56.7, 54.7, 42.1, 27.5, 24.1, 18.8.Example compound 5 is the early eluting enantiomer version of Examplecompound 4 after chiral separations as described above.Example compound 6 is the later eluting enantiomer version of Examplecompound 4 after chiral separations as described above.

Example compound 7 was synthesized according to Method 3 using3-trifluoromethyl-bromobenzene as the aryl bromide and N-Boc-pipecolicacid as the Boc-protected amino-acid. 2,4-Dichloro-5-fluoropyrimidinewas used as the chloro-heteroarene in Step 5b. ¹H NMR (500 MHz,DMSO-d6), δ (ppm): 8.24 (d, 1H, J=6.5 Hz), 7.66 (m, 1H), 7.63 (m, 3H),5.78 (t, 1H, J=4.2 Hz), 4.30 (dd, 1H, J=13.7, 3.7 Hz), 3.02 (ddd, 1H,J=14.7, 10.3, 4.7 Hz), 2.45 (dd, 1H, J=14.3, 3.7 Hz), 2.01 (dddd, 1H,J=14.1, 12.5, 5.5, 3.5 Hz), 1.64 (m, 3H), 1.41 (m, 1H). ¹³C NMR (125MHz, DMSO-d6), δ (ppm): 153.4 (d, J=27 Hz), 153.3 (d, J=30 Hz), 146.2(d, J=256 Hz), 145.0 (d, J=28 Hz), 141.2, 131.2, 130.4, 130.0 (q, J=31Hz), 124.7 (q, J=272 Hz), 124.2 (q, J=4 Hz), 123.5 (q, J=4 Hz), 55.9 (d,J=6 Hz), 42.7 (d, J=9 Hz), 28.3, 25.1, 19.3.Example compound 8 is the early eluting enantiomer version of Examplecompound 7 after chiral separations as described above.Example compound 9 is the later eluting enantiomer version of Examplecompound 7 after chiral separations as described above.

Example compound 121 was synthesized according to Method 3 using2-chloro-1-fluoro-4-iodobenzene as the aryl iodide and N-Boc-pipecolicacid as the Boc-protected amino-acid. 2,4-Dichloro-5-fluoropyrimidinewas used as the chloro-heteroarene in Step 5b. ¹H NMR (500 MHz,DMSO-d6), δ (ppm): 8.22 (d, 1H, J=6.6 Hz), 7.53 (ddd, 1H, J=7.1, 2.4,1.0 Hz), 7.41 (t, 1H, J=8.9 Hz), 7.32 (dddd, 1H, J=8.4, 4.7, 2.4, 1.0Hz), 5.70 (s, 1H), 4.27 (m, 1H), 3.02 (ddd, 1H, J=14.4, 11.1, 3.7 Hz),2.40 (m, 1H), 1.95 (dddd, 1H, J=14.2, 12.5, 5.4, 3.5 Hz), 1.62 (m, 3H),1.43 (m, 1H). ¹³C NMR (125 MHz, DMSO-d6), δ (ppm): 156.6 (d, J=246 Hz),153.3 (d, J=39 Hz), 153.3 (d, J=40 Hz), 146.2 (d, J=256 Hz), 145.0 (d,J=28 Hz), 137.5 (d, J=4 Hz), 129.2, 127.8 (d, J=7 Hz), 120.4 (d, J=18Hz), 117.6 (d, J=21 Hz), 55.3 (d, J=5 Hz), 42.6 (d, J=9 Hz), 28.3, 25.2,19.3.Example compound 10 is the early eluting enantiomer version of Examplecompound 121 after chiral separations as described above.Example compound 11 is the later eluting enantiomer version of Examplecompound 121 after chiral separations as described above.

Additional exemplary compounds are described in Tables 5-17.

TABLE 5 Example compounds prepared via Methods 1 and 3 with given corestructure. A “*” indicates a chiral compound. A “^(a)” indicates M + Naobserved in MS as major product. A “^(x)” indicates M—SO2 observed in MSas major product.

Sulfonyl derivatives of 6 Compound No. Expect M + H Observed M + H R 4444.00 444.00 4-Cl—Ph (racemate) 5 444.00 444.00 4-Cl—Ph* 6 444.00444.00 4-Cl—Ph* 12 424.06 424.10 4-Me—Ph 13 428.03 428.00 4-F—Ph 14410.04 410.00 Ph 15 374.04 374.00 cyclopropyl 16 348.03 348.00 Me 17445.00 445.00 2-Cl-pyrid-3-yl 314 461.99 461.96 2-F-4-Cl—Ph 315 461.99461.96 3-F-4-Cl—Ph 316 444.00 444.00 3-Cl—Ph 317 461.99 461.962-F-3-Cl—Ph 318 461.99 462.03 3-Cl-4-F—Ph 319 461.99 461.96 2-F-5-Cl—Ph320 479.98 480.02 2,4-DiF-5-Cl—Ph 321 458.02 458.02 4-Cl—Bn 322 445.00445.00 2-Cl-pyrid-5-yl 323 442.05  464.08^(a) 2-F—Bn 324 442.05 464.08^(a) 3-F—Bn 325 442.05  464.05^(a) 4-F—Bn 397 458.02  394.14^(x)2-Cl—Bn 398 458.02  480.04^(a) 3-Cl—Bn 399 410.02 410.023-Cl-propan-1-yl 483 414.05 414.08 1-Me-pyrazol-3-yl

TABLE 6 Example compounds prepared via Methods 1 and 3 with given corestructure. A “*” indicates a chiral compound. A “^(a)” indicates M + Naobserved in MS as major product. A “^(x)” indicates M—SO2 observed in MSas major product.

Sulfonyl derivatives of 18 Compound No. Expect M + H Observed M + H R 18404.07 404.05 4-Cl—Ph 19 370.11 370.10 Ph 20 420.12 420.10 2-Napthyl 21404.07 404.10 3-Cl—Ph 22 418.09  440.20^(a) 4-Cl—Bn 23 452.05 388.10^(x) 3,4-DiCl—Bn 24 418.09  354.20^(x) 2-Cl—Bn 25 422.06 422.104-Cl-2-F—Ph 26 406.09 406.10 2,5-DiF—Ph 27 384.12 384.10 3-Me—Ph 28422.06 422.10 3-Cl-2-F—Ph 426 384.12  406.14^(a) Bn

TABLE 7 Example compounds prepared via Method 4 with the given corestructure. A “*” indicates a chiral compound. A “^(a)” indicates M + Naobserved in MS as major product. A “^(x)” indicates M—SO2 observed in MSas major product.

Sulfonyl derivatives of 29 Compound No. Expect M + H Observed M + H R 29450.97 451.00 4-Cl—Ph 30 417.01 417.00 Ph 31 467.03 467.00 2-Napthyl 32450.97 451.00 3-Cl—Ph 33 464.99  401.10^(x) 4-Cl—Bn 34 498.95 521.00^(a) 3,4-DiCl—Bn 35 431.03  367.10^(x) Bn 36 464.99  401.10^(x)2-Cl—Bn 37 468.96 469.00 4-Cl-2-F—Ph 38 452.99 453.00 2-5-DiF—Ph 39447.02 447.00 4-OMe—Ph

TABLE 8 Example compounds prepared via Methods 1, 2 and 3 with givencore structure. A “*” indicates a chiral compound. A “^(a)” indicatesM + Na observed in MS as major product. A “^(x)” indicates M—SO2observed in MS as major product.

Sulfonamide aryls No. n Expect M + H Observed M + H R 3 2 405.07 405.106-CF3-pyrid-2-yl 4 2 444.00 444.00 3-Br-4-OMe—Ph (racemate) 5 2 444.00444.00 3-Br-4-OMe—Ph* 6 2 444.00 444.00 3-Br-4-OMe—Ph* 18 2 404.07404.05 3-CF3—Ph 40 0 415.97 416.00 3-Br-4-OMe—Ph 41 1 429.99 430.003-Br-4-OMe—Ph 42 2 444.00  466.10^(a) 4-Br-3-OMe—Ph 43 2 497.98 498.003-Br-4-OCF3—Ph 44 2 380.07  402.10^(a) benzo[d][1,3]diox-5-ole 45 2337.08 337.10 pyrid-2-yl 46 2 351.09 351.10 6-Me-pyrid-2-yl 47 2 362.07362.10 6-CN-pyrid-2-yl 48 2 421.06 421.10 6-OCF3-pyrid-2-yl 49 2 429.07429.00 3-CF3-4-CN—Ph 50 2 423.07 423.10 3-CF3-5-F—Ph 51 2 400.05 400.053-Cl-4-OMe—Ph 52 2 388.03 388.00 3-Cl-4-F—Ph 53 2 355.07 355.102-F-pyrid-5-yl 54 2 355.07 355.10 5-F-pyrid-2-yl 55 2 422.06 422.053-CF3-4-F—Ph 56 2 431.98 431.95 3-Br-4-F—Ph* 57 2 405.07 405.102-CF3-pyrid-4-yl 58 2 379.07 379.10 3-CN-4-F—Ph 59 2 355.07 355.102-F-pyrid-4-yl 60 2 431.98 432.00 3-Br-4-F—Ph 61 2 370.04 370.00 3-Cl—Ph62 2 355.07 355.10 4-F-pyrid-2-yl 63 2 406.06 406.10 6-CF3-pyrazin-2-yl64 2 438.03 438.00 3-CF3-4-Cl—Ph 65 2 405.07 405.10 5-CF3-pyrid-2-yl 662 378.09 378.05 2,3-dihydrobenzo-furan-5-yl 67 2 368.09 368.103-Me-4-F—Me—Ph 68 2 380.11 380.10 3-Me-4-OMe—Ph 69 2 438.03 438.003-CF3-4-Cl—Ph* 70 2 428.01 428.00 3-Br-4-Me—Ph 71 2 431.98 431.953-F-4-Br—Ph 72 2 422.06 422.10 3-CF3-5-F—Ph 73 2 406.06 406.104-CF3-2-pyrimid-2-yl 74 2 447.95 470.00 3-Br-4-Cl—Ph 75 2 372.06 372.103,4-DiF—Ph 76 2 370.04 370.10 4-Cl—Ph 77 2 351.09 351.10 2-Me-pyrid-4-yl78 2 354.07 354.10 4-F—Ph 79 2 405.07 405.10 4-CF3-pyrid-2-yl 80 2362.07 362.10 2-CN-pyrid-4-yl 81 2 395.08 395.10 (2-(methylCarboxylate)- pyrid-4-yl 82 2 355.07 355.10 5-F-pyrid-3-yl 83 2 405.07405.10 5-CF3-pyrid-3-yl 84 3 458.02 458.00 3-Br-4-OMe—Ph 220 2 438.03438.00 3-CF3-4-Cl—Ph* 427 2 390.05 390.09 3,4,5-TriF—Ph 446 2 431.98431.98 3-Br-4-F—Ph*

TABLE 9 Example compounds prepared via Methods 3, 5 and 6 with givencore structure. A “*” indicates a chiral compound. A “^(a)” indicatesM + Na observed in MS as major product. A “^(x)” indicates M—SO2observed in MS as major product.

substituted sulfonyl pyrrolidines Compound Expected Observed Number M +H M + H R1 R2 R3 85 465.97 465.95 3-Br-4-OMe—Ph F, F H, H 86 497.98497.95 3-Br-4-OMe—Ph H, H H, CF₃ 87 438.03 438.00 3-CF₃-4-Cl—Ph H, Me H,H 88 370.04 370.00 4-Cl—Ph H, Me H, H 89 404.07 404.10 3-CF₃—Ph H, Me H,H 90 497.98 497.95 3-Br-4-OMe—Ph H, CF₃ H, H 91 378.09 378.102,3-dihydro-benzofuran-5-yl H, Me H, H 92 431.98 432.00 3-F-4-Br—Ph H,Me H, H 93 379.07 379.10 3-CN-4-F—Ph H, Me H, H 94 497.98 498.003-Br-4-OCF₃—Ph H, Me H, H 95 354.07 354.10 4-F—Ph H, Me H, H 96 380.07380.10 benzo[d][1,3]diox-5-ole H, Me H, H 97 372.06 372.10 3,4-DiF—Ph H,Me H, H 98 390.05 390.10 2,3,4-DiF—Ph H, Me H, H 99 447.95 448.003-Br-4-Cl—Ph H, Me H, H 100 422.05 422.10 3-CF₃-4-F—Ph H, Me H, H 101368.08 368.10 3-Me-4-F—Ph H, Me H, H 102 444.00 444.00 3-Br-4-OMe—Ph H,Me H, H 103 515.96 516.00 3-Br-4-OMe—Ph F, CF₃ H, H 104 428.00 428.003-Br-4-Me—Ph H, Me H, H 105 388.03 388.00 3-Cl-4-F—Ph H, Me H, H 106431.98 432.00 3-Br-4-F—Ph H, Me H, H 107 370.04 370.00 3-Cl—Ph H, Me H,H 108 497.98 497.95 3-Br-4-OMe—Ph* H, CF₃ H, H 109 497.98 497.953-Br4-OMe—Ph* H, H H, CF₃ 110 465.97 465.95 3-Br-4-OMe—Ph* F, F H, H 111497.97 497.95 3-Br4-OMe—Ph* H, H H, CF₃ 112 447.97 447.95 3-Br4-OMe—Ph*H, H H, F 113 465.96 487.95 3-Br-4-OMe—Ph F, F H, H 41 429.98 430.003-Br-4-OMe—Ph H, H H, H 114 447.97 470.00 3-Br4-OMe—Ph* H, H H, F 115447.97 447.95 3-Br4-OMe—Ph H, F H, H 116 465.96  488.00^(a)3-Br-4-OMe—Ph* F, F H, H 117 497.97 497.95 3-Br-4-OMe—Ph* H, CF₃ H, H118 444.00 444.00 3-Br-4-OMe—Ph* H, H H, Me 119 497.98 497.953-Br-4-OMe—Ph* H, H H, CF₃ 120 497.98 497.95 3-Br-4-OMe—Ph* H, H H, CF₃

TABLE 10 Example compounds prepared via Methods 1, 2 and 3 with givencore structure. A “*” indicates a chiral compound.

Aryl Derivatives of 7 Compound No. Expected M + H Observed M + H R 7360.09 360.10 3-CF3—Ph 8 360.09 360.10 3-CF3—Ph* 9 360.09 360.053-CF3—Ph* 10 344.05 344.10 3-Cl-4-F—Ph* 11 344.05 344.05 3-Cl-4-F—Ph*121 344.05 344.05 3-Cl-4-F—Ph 122 378.08 378.05 3-CF3-5-F—Ph 123 361.08361.05 6-CF3-pyrid-2-yl 124 388.00 387.95 3-Br-4-F—Ph 125 403.97 403.953-Br-4-Cl—Ph* 126 453.99 453.95 3-Br-4-OCF3—Ph 127 310.09 310.05 4-F—Ph128 394.05 394.00 3-CF3-4-Cl—Ph 129 328.08 328.05 3,4-DiF—Ph 130 324.11324.10 3-Me-4-F—Ph 131 377.08 377.05 6-OCF3-pyrid-2-yl 132 400.02 400.003-Br-4-OMe—Ph 133 311.09 311.05 6-F-pyrid-2-yl 134 374.10 374.102-Me-5-CF3—Ph 135 374.10 374.10 2-Me-3-CF3—Ph 274 362.08 362.126-CF3-pyrazin-2-yl 275 323.11 323.11 2-OMe-pyrid-4-yl 276 327.06 327.096-Cl-pyrid-2-yl 277 328.05 328.05 6-Cl-pyrazin-2-yl 278 361.08 361.084-CF3-pyrid-2-yl 279 349.09 349.05 2(3H)-benzoxazolon-6-yl 280 327.06327.06 5-Cl-pyrid-3-yl 281 384.03 384.06 3-Br-4-Me—Ph 283 343.11 343.084-quinoline 284 332.11 332.07 imidazo[1,2-a]pyrid-5-yl 285 326.06 326.033-Cl—Ph 286 343.11 343.15 isoquinolin-8-yl 287 438.00 437.963-CF3-4-Br—Ph 288 400.02 400.06 3-OMe-4-Br—Ph 289 390.10 390.103-CF3-4-OMe—Ph 290 388.00 387.97 3-F-4-Br—Ph 291 333.10 333.10[1,4,5]triazolo[1,2-a]pyridin-6-yl 292 332.11 332.11imidazo[1,2-a]pyrid-4-yl 294 352.12 352.09 3,4-DiOMe—Ph 297 311.09311.05 2-F-pyrid-5-yl 298 342.10 342.06 3-DiFluoroMethyl—Ph 299 360.09360.05 3-DiFluoroMethyl-4-F—Ph 300 414.11 414.075-CF3-1-Me-Benzimidazol-7-yl 326 343.11 343.15 6-quinoline 327 346.07346.07 3,4,5-TriF—Ph 328 327.06 327.09 2-Cl-pyrid-4-yl 329 293.10 293.06pyrid-2-yl 330 307.11 307.11 5-Me-pyrid-3-yl 331 360.02 359.993,4,-DiCl—Ph 332 361.08 361.08 4-CF3-pyrid-3-yl 333 333.10 333.07[1,4,5]triazolo[1,2-a]pyridin-7-yl 334 332.11 332.11imidazo[1,5-a]pyrid-6-yl 335 311.09 311.12 2-F-pyrid-3-yl 336 332.11332.11 imidazo[1,2-a]pyrid-3-yl 337 333.10 333.14imidazo[1,2-a]pyridizin-3-yl 338 333.10 333.07imidazo[1,2-a]pyrizin-3-yl 339 333.10 333.14imidazo[1,2-a]pyridizin-5-yl 340 349.07 349.10 benzthiazol-2-yl 341349.07 349.07 benzthiazol-6-yl 343 344.05 344.02 3-Cl-4-F—Ph* 345 394.05394.09 3-CF3-4-Cl—Ph* 346 344.05 344.02 3-Cl-4-F—Ph* 348 394.05 394.093-CF3-4-Cl—Ph* 352 400.10 400.13 7-CF3-1H-Indazol-5-yl 353 378.08 378.082-F-5-CF3—Ph 354 455.99 455.95 2-Br-3-F-6-CF3—Ph 355 378.08 378.082-F-3-CF3—Ph 357 346.04 346.08 2-Cl-5-F-pyrimid-4-yl 358 298.09 298.053-Me-1,2,4-oxadiazol-5-yl 362 292.10 292.07 Ph 363 298.09 298.125-Me-1,3,4-oxadiazol-2-yl 364 297.09 297.13 5-Me-oxazol-2-yl 366 297.09297.09 4-Me-oxazol-2-yl 369 344.05 344.05 2-F-3-Cl—Ph 370 311.09 311.055-F-pyrid-3-yl 371 334.11 334.11 2,3-dihydrobenzofuran-5-yl 372 311.09311.05 5-F-pyrid-2-yl 373 293.10 293.06 pyrid-3-yl 374 333.10 333.10[1,4,5]triazolo[1,2-a]pyridin-5-yl 375 311.09 311.09 3-F-pyrid-4-yl 376361.08 361.08 5-CF3-pyrid-3-yl 377 308.11 308.14 2-Me-pyrimid-4-yl 378294.09 294.13 pyrimid-5-yl 379 358.06 358.10 4-Cl-5-OMe-pyrimid-2-yl 380323.11 323.11 2-OMe-pyrid-3-yl 388 345.05 345.08 3-F-6-Cl-pyrid-2-yl 389333.10 333.07 [1,3,4]triazolo[4,5-a]pyridin-5-yl 390 345.05 345.082-F-5-Cl-pyrid-3-yl 391 294.09 294.13 pyrimid-4-yl 392 342.07 342.102-Me-5-Cl-pyrimid-4-yl 393 359.11 359.14 3-Hydroxy-quinolin-6-yl 400344.05 344.09 2-F-5-Cl—Ph 404 344.05 344.02 3-Cl-5-F—Ph* 407 307.11307.08 2-Me-Pyrid-3-yl 408 327.06 327.02 4-Cl-pyrid-2-yl 409 298.09298.12 5-Me-1,2,4-oxadiazol-3-yl 410 361.08 361.05 2-CF3-pyrid-4-yl 411307.11 307.08 2-Me-pyrid-5-yl 412 311.09 311.09 4-F-pyrid-2-yl 413361.08 361.05 2-CF3-pyrid-5-yl 414 307.11 307.15 6-Me-pyrid-2-yl 417400.10 400.10 7-CF3-1H-benzimidazol-5-yl 420 344.05 344.02 3-Cl-5-F—Ph*421 332.11 332.07 imidazo[1,2-a]pyrid-6-yl 422 378.08 378.083-CF3-4-F—Ph* 423 378.08 378.08 3-CF3-5-F—Ph* 429 260.06 260.02 Carboxy

TABLE 11 Example compounds prepared via Method 3 with given corestructure. A “*” indicates a chiral compound.

bicyclic derivatives Expect Obs. No. M + H M + H R1 R2 136 400.00 399.953-Br-4-F—Ph 2-Cl-5-F-pyrimidin-4-yl 137 373.08 373.05 6-CF₃-pyrid-2-yl2-Cl-5-F-pyrimidin-4-yl 138 412.02 412.05 3-Br-4-OMe—Ph2-Cl-5-F-pyrimidin-4-yl 139 390.08 390.05 3-CF₃-5-F—Ph2-Cl-5-F-pyrimidin-4-yl 216 402.10 402.10 3-CF₃-5-F—Ph 3-Cl-6-F—Bn 217368.08 368.05 3-Cl-4-F—Ph 3-Cl-6-F—Bn 430 372.09 372.05 3-CF3—Ph2-Cl-5-F-pyrimidin-4-yl 431 356.05 356.02 3-Cl-4-F—Ph2-Cl-5-F-pyrimidin-4-yl 444 385.11 385.15 6-CF3-pyrid-2-yl 3-Cl-6-F—Bn

TABLE 12 Example compounds prepared via Methods 1, 2 and 3 with givencore structure. A “*” indicates a chiral compound.

Aryl Derivatives of 1 No. Exp M + H Obs M + H R1 R2 1 339.08 339.106-Cl-pyrid-2-yl 3-Cl-4-F—Ph 2 373.11 373.10 2-F-5-Cl—Ph 6-CF3-pyrid-2-yl140 354.12 354.10 4-Cl—Ph 3-CF3—Ph 141 364.16 364.15 2-F-5-Cl—Ph6-(2-amino)ethan-1- ol)pyridin-2-yl) 142 348.16 348.15 2-F-5-Cl—Ph6-(2-amino)eth-ane)pyridin- 2-yl) 143 356.11 356.10 5-Cl-pyrid-3-yl6-CF3-pyrid-2-yl 144 346.12 346.10 2-F-5-Cl—Ph 6-azido-pyrid-2-yl 145339.08 339.05 4-Cl-pyrid-2-yl 3-Cl-4-F—Ph 146 355.12 355.10 4-Cl—Ph6-CF3-pyrid-2-yl 147 339.08 339.05 5-Cl-pyrid-3-yl 3-Cl-4-F—Ph 148416.06 416.05 3-Br—Ph 3-CF3-5-F—Ph 149 348.16 348.15 2-F-5-Cl—Ph6-N(Me)2-pyrid-2-yl 150 406.08 406.05 2-F-5-Cl—Ph 3-CF3-4-Cl—Ph 151372.05 372.00 3,5-diCl—Ph 3-Cl-4-F—Ph 152 339.15 339.10 3-F—Ph6-CF3-pyrid-2-yl 153 389.08 389.05 3,4-diCl—Ph 6-CF3-pyrid-2-yl 154354.12 354.10 3-Cl—Ph 3-CF3—Ph 155 356.08 356.05 3-Cl-5-F—Ph 3-Cl-4-F—Ph156 351.17 351.15 3-OMe—Ph 6-CF3-pyrid-2-yl 157 372.11 372.10 3-Cl—Ph3-CF3-5-F—Ph 158 372.05 372.00 2,5-diCl—Ph 3-Cl-4-F—Ph 159 389.08 389.053,5-diCl—Ph 6-CF3-pyrid-2-yl 160 356.08 356.05 2-F-5-Cl—Ph 3-Cl-4-F—Ph161 373.11 373.10 3-Cl-4-F—Ph 6-CF3-pyrid-2-yl 162 356.11 356.104-Cl-pyrid-2-yl 6-CF3-pyrid-2-yl 163 322.12 322.10 2-F-5-Cl—Ph 4-F—Ph164 373.11 373.10 3-Cl-5-F—Ph 6-CF3-pyrid-2-yl 165 356.11 356.106-Cl-pyrid-2-yl 6-CF3-pyrid-2-yl 166 356.11 356.10 2-Cl-pyrid-4-yl6-CF3-pyrid-2-yl 167 390.10 390.10 2-F-5-Cl—Ph 3-CF3-5-F—Ph 168 334.15334.10 2-F-5-Cl—Ph 6-NHMe-pyrid-2-yl 169 389.10 389.10 2-F-5-Cl—Ph6-OCF3-pyrid-2-yl 170 355.12 355.10 3-Cl—Ph 6-CF3-pyrid-2-yl 171 323.11323.10 2-F-5-Cl—Ph 6-F-pyrid-2-yl 172 373.11 373.10 2-F-3-Cl—Ph6-CF3-pyrid-2-yl 173 399.07 399.05 3-Br—Ph 6-CF3-pyrid-2-yl 174 339.08339.05 2-Cl-pyrid-4-yl 3-Cl-4-F—Ph 175 389.15 389.10 3-CF3—Ph6-CF3-pyrid-2-yl 176 389.08 389.05 2,5-diCl—Ph 6-CF3-pyrid-2-yl 245412.05 412.05 2-F-3-Cl—Bn 3-Br-4-OMe—Ph 432 412.05 412.05 2-F-5-Cl—Bn3-Br-4-OMe—Ph 433 400.03 400.06 2-F-5-Cl—Bn 3-Br-4-F—Ph 434 340.11340.07 2-F-5-Cl—Bn 3,4-DiF—Ph 435 336.13 336.10 2-F-5-Cl—Bn 3-Me-4-F—Ph436 466.02 465.98 2-F-5-Cl—Bn 3-Br-4-OCF3—Ph 437 416.00 416.032-F-5-Cl—Bn 3-Br-4-Cl—Ph 438 364.16 364.12 2-F-5-Cl—Bn 6-(N,O-dimethylhydroxylamine)-pyridin-2-yl) 439 402.14 402.17 2-F-5-Cl—Bn6-(N-(2,2,2-trifluoroethyl))- pyridin-2-yl)

TABLE 13 Example compounds prepared via Method 3 with given corestructure. Stereochemistry at bicycle is (2S,6R). If designated as *,then stereochemistry at bicycle is (2R,6S).

Sulfonyl bicyclic derivatives No. Expect M + H Observed M + H R1 R2 177456.01 456.00 4-Cl—Ph 3-Br-4-OMe—Ph 178 443.99 443.95 4-Cl—Ph3-Br-4-F—Ph 179 443.99 443.95 4-Cl—Ph 3-Br-4-F—Ph* 180 443.99 443.953-Cl—Ph 3-Br-4-F—Ph 181 491.96 514.10 3,4-DiCl—Bn 3-Br-4-F—Ph 182 424.04360.10 Bn 3-Br-4-F—Ph 183 458.00 394.10 2-Cl—Bn 3-Br-4-F—Ph 184 461.98461.95 2-F-4-Cl—Ph 3-Br-4-F—Ph

TABLE 14 Example compounds prepared via Method 3 with given corestructure. A “*” indicates a chiral compound.

Di-Hetero derivatives No. Expect M + H Obs M + H R1 R2 X 185 433.97433.95 4-Cl-PhSO2 3-Br-4-F—Ph O 186 363.07 363.05 2-Cl-5-F-6-CF3-pyrid-2- O pyrimidin-4-yl yl 187 402.00 401.95 2-Cl-5-F-3-Br-4-OMe— O pyrimidin-4-yl Ph 188 362.07 362.05 2-Cl-5-F- 3-CF3—Ph Opyrimidin-4-yl 189 346.03 346.00 2-Cl-5-F- 3-Cl-4-F—Ph O pyrimidin-4-yl190 380.06 380.05 2-Cl-5-F- 3-CF3-5-F—Ph O pyrimidin-4-yl 191 375.09375.05 2-F-5-Cl—Bn 6-CF3-pyrid-2- O yl 192 414.03 414.00 2-F-5-Cl—Bn3-Br-4-OMe— O Ph 193 374.10 374.05 2-F-5-Cl—Bn 3-CF3—Ph O 194 358.06358.05 2-F-5-Cl—Bn 3-Cl-4-F—Ph O 195 392.09 392.05 2-F-5-Cl—Bn3-CF3-5-F—Ph O 222 445.98 445.95 4-Cl—PhSO2 3-Br-4-OMe— O Ph 282 375.10375.06 2-Cl-5-F- 3-CF3—Ph NMe Pyrimid-4-yl 301 396.04 396.00 2-Cl-5-F-3-CF3-5-F—Ph S pyrimid-4-yl 302 412.03 412.07 2-Cl-5-F- 3-CF3-5-F—Ph SOpyrimid-4-yl 303 428.03 427.99 2-Cl-5-F- 3-CF3-5-F—Ph SO2 pyrimid-4-yl305 393.09 393.05 2-Cl-5-F- 3-CF3-5-F—Ph NMe pyrimid-4-yl 306 464.13464.13 2-Cl-5-F- 3-CF3-5-F—Ph NCO(CH2N(Me)2) pyrimid-4-yl 307 465.11465.08 2-Cl-5-F- 3-CF3-5-F—Ph NCO(CMe2OH) pyrimid-4-yl 308 421.09 421.052-Cl-5-F- 3-CF3-5-F—Ph NAc pyrimid-4-yl 309 457.07 457.02 2-Cl-5-F-3-CF3-5-F—Ph NSO2Me pyrimid-4-yl 344 380.06 380.09 2-Cl-5-F-3-CF3-5-F—Ph* O pyrimidin-4-yl 347 380.06 380.06 2-Cl-5-F- 3-CF3-5-F—Ph*O pyrimidin-4-yl 419 375.12 375.12 2-OMe-5-F- 3-CF3-5-F—Ph NHpyrimidin-4-yl 456 378.05 378.05 2-Cl-5-F- 3-CF3—Ph S pyrimid-4-yl 460362.01 362.01 2-Cl-5-F- 3-Cl-4-F—Ph S pyrimid-4-yl

TABLE 15 Example compounds prepared via Method 3 with given corestructure.

substituted derivatives Exp Obs Number M + H M + H a b R1 R2 R3 196396.07 396.05 1 1 3-CF3-5-F—Ph F H 197 378.08 378.05 1 1 3-CF3—Ph F H198 414.06 414.05 1 1 3-CF3-5-F—Ph F F 199 396.07 396.05 1 1 3-CF3—Ph FF 200 390.08 390.05 1 1 3-CF3-5-F—Ph CH2 null 201 394.08 394.05 1 13-CF3-5-F—Ph OH H 202 376.09 376.05 1 1 3-CF3—Ph OH H 223 366.02 366.000 1 3-CF3-5-F—Ph F F 224 400.05 400.00 0 1 3-Cl-4-F—Ph F F 221 361.08361.08 0 1 6-CF3-pyrid-2-yl* Me H 237 400.02 400.06 0 1 3-Br-4-Ome—Ph MeH 238 392.06 392.06 1 1 3-CF3-5-F—Ph O null 239 374.07 374.07 1 13-CF3—Ph O null 240 401.98 401.95 1 1 3-Br-4-F—Ph O null 241 404.00404.03 1 1 3-Br-4-F—Ph OH H 244 385.99 386.02 CH 1 3-Br-4-F—Ph H null246 423.98 423.95 1 1 3-Br-4-F—Ph F F 247 402.14 402.17 2 0 3-CF3—PhPropyl H 248 428.05 428.05 2 0 3-Br-4-Ome—Ph Ethyl H 249 414.04 414.00 20 3-Br-4-Ome—Ph Methyl H 250 428.05 428.05 2 0 3-Br-4-Ome—Ph Ethyl H(syn) 251 428.05 428.05 2 0 3-Br-4-Ome—Ph Ethyl H (anti) 252 392.10392.13 1 2 3-CF3-5-F—Ph H H 253 375.10 375.06 1 2 6-CF3-pyrid-2-yl H H273 359.03 358.99 CONH 1 3-Cl-4-F—Ph H H 293 360.05 360.01 1 13-Cl-4-F—Ph OH H 304 507.16 507.12 0 2 3-CF3-5-F—Ph O-Ethyl- HMorpholine 310 394.07 394.07 0 2 3-CF3-5-F—Ph (Anti) H OH 311 394.07394.07 0 2 3-CF3-5-F—Ph (syn) OH H 312 396.07 396.07 0 2 3-CF3-5-F—Ph(Anti) H F 313 396.07 396.07 0 2 3-CF3-5-F—Ph (syn) F H 342 407.11407.11 1 1 3-CF3-5-F—Ph NHMe H 367 375.06 375.06 1 CONH 3-Cl-4-F—Ph H H383 402.10 402.14 2 0 3-CF3—Ph CH2OCH2 384 390.10 390.06 0 2 3-CF3—PhOMe H 385 418.01 418.05 0 2 3-F-4-Br—Ph OMe H 386 402.10 402.10 0 23-CF3—Ph CH2OCH2 387 430.01 430.01 0 2 3-F-4-Br—Ph CH2OCH2 394 372.11372.07 2 0 3-CF3—Ph (anti) H OMe 416 390.10 390.10 2 0 3-CF3—Ph (syn)OMe H 418 421.12 421.12 0 2 3-F-5-CF3—Ph N,N-DiMe H 424 420.13 420.13 20 3-CF3—Ph Propyl F 448 360.09 360.09 0 1 3-CF3—Ph Me H 450 378.08378.04 0 1 3-CF3-5-F—Ph Me H 457 432.05 432.02 0 1 3-CF3-5-F—Ph CF3 H464 348.03 348.06 0 1 3-Cl-4-F—Ph F H 466 421.99 421.99 0 13-Br-4-OMe—Ph F F 467 382.05 382.05 0 1 3-CF3—Ph F F 468 382.05 382.02 01 3-CF3-5-F—Ph F H 473 414.06 414.06 0 1 3-CF3—Ph CF3 H 476 398.03397.99 0 1 3-Cl-4-F—Ph CF3 H 481 365.06 365.10 0 1 6-CF3-pyrid-2-yl F H482 383.05 383.01 0 1 6-CF3-pyrid-2-yl F F

TABLE 16 Example compounds prepared via Method 4 with given corestructure.

3-substituted piperidines Number Exp M + H Obs M + H R1 R2 R3 203 401.04401.00 3-Cl—Bn 3-Br-4-F—Ph D 204 373.12 373.10 3-Cl—Bn 3-CF₃—Ph D 205391.11 391.10 3-F-3-Cl—Bn 3-CF₃—Ph D 206 417.07 417.05 3-Br—Bn 3-CF₃—PhD 207 379.09 379.05 2-Cl-5-F-pyrimidin-4-yl 3-CF₃—Ph D 208 379.05 379.082-Cl-5-F-pyrimidin-4-yl 3-CF₃—Ph (*1) D 209 379.05 379.082-Cl-5-F-pyrimidin-4-yl 3-CF₃—Ph (*2) D 210 406.00 405.952-Cl-5-F-pyrimidin-4-yl 3-Br-4-F—Ph H 211 405.95 405.992-Cl-5-F-pyrimidin-4-yl 3-Br-4-F—Ph (syn) H 212 418.00 418.022-Cl-5-F-pyrimidin-4-yl 3-Br-4-OMe—Ph H 213 418.00 418.022-Cl-5-F-pyrimidin-4-yl 3-Br-4-OMe—Ph (syn) H 214 406.95 407.002-Cl-5-F-pyrimidin-4-yl 3-Br-4-F—Ph (syn) D 219 461.95 461.994-Cl—Ph-Sulfonyl 3-Br-4-OMe—Ph H 440 391.11 391.11 2-F-5-Cl—Bn 3-CF3—PhD 441 391.11 391.15 3-Cl-5-F—Bn 3-CF3—Ph D 442 407.08 407.05 2,5-diCl—Bn3-CF3—Ph D *1 indicates earlier eluting enantiomer. *2 indicates latereluting enantiomer. *3 indicates transdiasteroemeric relationshipbetween R2 and F.

TABLE 17 Example compounds prepared via Method 1, steps 5b-5d with givencore structure. A “*” indicates a compound made via Method 8.

N-aryl derivatives Compound Expect Observed No. M + H M + H R1 R2 132400.02 400.00 2-Cl-5-F-pyrimid-4-yl 3-Br-4-OMe—Ph 215 399.03 399.002-Cl-4-F-pyridin-6-yl 3-Br-4-OMe—Ph 218 314.11 314.10 3-CN—Ph*3-Cl-4-F—Ph 225 396.08 396.07 2-OMe-5-F-pyrimidin-4-yl 3-Br-4-OMe—Ph 226382.04 382.03 2-Cl-pyrimidin-4-yl 3-Br-4-OMe—Ph 227 415.07 415.062-CF3-pyridin-6-yl 3-Br-4-OMe—Ph 228 400.02 400.026-Cl-5-F-pyrimidin-4-yl 3-Br-4-OMe—Ph 229 382.04 382.036-Cl-pyrizin-2-yl 3-Br-4-OMe—Ph 230 416.06 416.06 6-CF3-pyrizin-2-yl3-Br-4-OMe—Ph 231 424.99 424.99 5-Br-pyridin-2-yl 3-Br-4-OMe—Ph 232415.07 415.06 5-CF3-pyridin-2-yl 3-Br-4-OMe—Ph 233 366.07 366.065-F-pyrimidin-4-yl 3-Br-4-OMe—Ph 234 396.05 396.052-Cl-5-Me-pyrimidin-4-yl 3-Br-4-OMe—Ph 235 412.05 412.042-Cl-5-OMe-pyrimidin-4-yl 3-Br-4-OMe—Ph 236 396.05 396.056-Cl-5-Me-pyrimidin-2-yl 3-Br-4-OMe—Ph 242 320.17 320.202-N,N-DiMe-5-F-pyrimid-4-yl 6-F-pyrid-2-yl 243 345.22 345.262-N,N-DiMe-5-F-pyrimid-4-yl 6-N,N-DiMe-pyrid- 2-yl 254 397.20 397.172-N,N-DiEt-5-F-pyrimid-4-yl 3-CF3—Ph* 255 411.18 411.182-morpholino-5-F-pyrimid-4-yl 3-CF3—Ph* 256 397.17 397.202-N-(3-hydroxyazetidine)-5-F- 3-CF3—Ph* pyrimid-4-yl 257 342.10 342.132-Cl-pyrimid-4-yl 3-CF3—Ph 258 342.10 342.13 4-Cl-pyrimid-2-yl 3-CF3—Ph259 293.10 293.10 2-Cl-pyrimid-4-yl 6-F-pyrid-2-yl 260 293.10 293.134-Cl-pyrimid-2-yl 6-F-pyrid-2-yl 261 359.09 359.06 2-Cl-5-F-pyrid-4-yl3-CF3—Ph 262 310.09 310.06 2-Cl-5-F-pyrid-4-yl 6-F-pyrid-2-yl 263 359.09359.13 3-F-6-Cl-pyrid-2-yl 3-CF3—Ph 264 310.09 310.063-F-6-Cl-pyrid-2-yl 6-F-pyrid-2-yl 265 356.11 356.082-Cl-5-Me-pyrimidin-4-yl 3-CF3—Ph 266 356.11 356.154-Cl-5-Me-pyrimidin-2-yl 3-CF3—Ph 267 307.11 307.112-Cl-5-Me-pyrimidin-4-yl 6-F-pyrid-2-yl 268 307.11 307.154-Cl-5-Me-pyrimidin-2-yl 6-F-pyrid-2-yl 269 340.14 340.142-Me-5-F-pyrimidin-4-yl 3-CF3—Ph 270 291.14 291.112-Me-5-F-pyrimidin-4-yl 6-F-pyrid-2-yl 271 356.14 356.102-OMe-5-F-pyrimidin-4-yl 3-CF3—Ph 272 307.14 307.142-OMe-5-F-pyrimidin-4-yl 6-F-pyrid-2-yl 295 356.14 356.172-OMe-4-F-Pyrimid-5-yl 3-CF3—Ph 296 307.14 307.10 2-OMe-4-F-Pyrimid-5-yl6-F-pyrid-2-yl 349 342.12 342.12 2-hydroxy-5-F-Pyrimid-4-yl 3-CF3—Ph 350342.12 342.16 2-hydroxy-5-F-Pyrimid-4-yl 3-CF3—Ph 351 360.09 360.055-F-6-Cl-pyrimid-4-yl 3-CF3—Ph 356 342.12 342.166-hydroxy-5-F-Pyrimid-4-yl 3-CF3—Ph 359 341.10 341.07 2-Cl-pyrid-4-yl3-CF3—Ph 360 342.10 342.10 6-Cl-pyrimid-4-yl 3-CF3—Ph 361 372.11 372.112-Cl-5-OMe-pyrimid-4-yl 3-CF3—Ph 365 360.09 360.05 2-Cl-pyrimid-4-yl2-F-3-CF3—Ph 368 344.12 344.08 2,5-Di-F-Pyrimid-4-yl 3-CF3—Ph 381 307.11307.08 3-F-6-Cl-5-Pyrid-2-yl 2-Me-primid-4-yl 382 293.10 293.103-F-6-Cl-5-Pyrid-2-yl pyrimid-5-yl 395 327.06 327.023-F-6-Cl-6-Pyrid-2-yl 6-Cl-Pyrazin-2-yl 396 290.12 290.122-Me-pyrimid-4-yl 6-Cl-Pyrazin-2-yl 401 324.13 324.132-hydroxy-pyrimid-4-yl 3-CF3—Ph 402 359.09 359.13 2-Cl-3-F-pyrid-4-yl3-CF3—Ph 403 360.09 360.05 2-Cl-pyrimid-4-yl 2-F-5-CF3—Ph 405 326.06326.06 2-Cl-pyrimid-4-yl 2-F-3-Cl—Ph 406 326.06 326.03 2-Cl-pyrimid-4-yl2-F-5-Cl—Ph 415 360.09 360.09 6-Cl-3-F-pyrid-2-yl 2-CF3-5-pyrid-5-yl 443324.07 324.07 3-Cl—Ph* 3-Cl-4-F—Ph 447 346.14 346.102,3-dihydro-1H-inden-2-ol-1-yl 3-Cl-4-F—Ph 449 424.02 424.022-Cl-3-CN-5-F-pyrid-6-yl 3-Br-4-OMe—Ph 451 331.14 331.14 3-ethynyl—Ph6-CF3-pyrid-2-yl 452 375.13 375.13 4-CF3—Ph* 6-CF3-pyrid-2-yl 453 358.10358.06 4-CF3—Ph* 3-Cl-4-F—Ph 454 341.10 341.10 4-Cl—Ph* 6-CF3-pyrid-2-yl455 337.15 337.19 3-OMe—Ph* 6-CF3-pyrid-2-yl 458 304.13 304.09 4-Me—Ph*3-Cl-4-F—Ph 459 341.10 341.10 3-Cl—Ph* 6-CF3-pyrid-2-yl 461 340.10340.07 2-OMe-5-F-pyrimidin-4-yl 3-Cl-4-F—Ph 462 331.11 331.154-azido—Ph* 3-Cl-4-F—Ph 463 314.11 314.15 2-ethynyl—Ph* 3-Cl-4-F—Ph 465320.12 320.09 3-OMe—Ph* 3-Cl-4-F—Ph 469 343.09 343.06 2-Cl-pyrimid-4-yl6-CF3-pyrid-2-yl 470 326.06 326.03 2-Cl-pyrimid-4-yl 3-Cl-4-F—Ph 471318.14 318.18 3,5-DiMe—Ph* 3-Cl-4-F—Ph 474 337.15 337.19 4-OMe—Ph*6-CF3-pyrid-2-yl 475 308.10 308.10 4-F—Ph* 3-Cl-4-F—Ph 477 348.12 348.08methl 4-benzoate* 3-Cl-4-F—Ph 478 365.15 365.11 methl 4-benzoate*6-CF3-pyrid-2-yl 479 376.16 376.16 2-morpholino-pyrid-3-yl* 3-Cl-4-F—Ph480 306.12 306.12 2-Me-pyrimid-4-yl 3-Cl-4-F—Ph

Methods for Providing Pain Relief by Administration of MOR PAMs

Although agonists that are highly selective with respect to thedifferent opioid receptor types exist, they are still beset by numerousside effects. Many of the side effects of these receptor-selectiveagonists are not due to off-target effects, but result fromindiscriminate activation all receptors throughout the body; therebyactivating MORs in tissues or regions of the CNS in which receptoractivation is undesirable (i.e., areas in which pain is not beingexperienced). Such side effects are unlikely to be addressed by thedevelopment of more highly selective agonists.

Thus, one advantage of the disclosed methods and compositions is thecapability of MOR PAMs, when administered in vivo in the absence of anexogenous orthosteric MOR ligand, to selectively increase receptoractivity only in regions where endogenous agonists are present, therebypreserving the termporally- and spatially-limited nature of theendogenous opioid response. This property of MOR-PAMs will result infewer “on-target” (i.e., MOR-mediated) side effects compared to the useof a MOR agonist.

Another advantage of the MOR PAMs disclosed herein is that their useavoids the sustained receptor activation that ensues betweenadministration and clearance of an exogenous opioid. Thus, the use ofMOR PAMs will minimize compensatory mechanisms, such as receptordownregulation and desensitization, that can lead to tolerance and/ordependence.

Because of the known basal activity of endogenous opioids (Roques et al.(2012) Nat. Rev. Drug Discov. 11:292-310; Levine et al. (1978) Nature272:826-827), administration of a MOR PAM, in the absence of anexogenous opioid, will provide an analgesic effect, by increasing thebaseline activity of the endogenous opioid. See example 9.

The MOR PAM compounds described herein are useful for treatment ofclinical acute pain, inflammatory pain, and for a variety of other uses.Alternatively, in a chronic pain situation, it is known that there aretemporal and spatial relationships between the physiological releases ofendogenous MOR ligands in inflamed and non-inflamed tissues. Thus, theMOR PAMs disclosed herein additionally provide methods for chronic painstudies wherein the pharmacodynamic measurements are designed such thatpain measurement post onset of chronic pain is decreased, and/or thereis a decrease and/or delay in the intensity of the development ofchronic pain and/or a delay in the onset of chronic pain.

Pharmaceutical Compositions and Formulations

Various pharmaceutical compositions and techniques for their preparationand use are known to those of skill in the art in light of the presentdisclosure. For a detailed listing of suitable pharmacologicalcompositions and techniques for their administration one may refer totexts such as Remington's Pharmaceutical Sciences, 17th ed. 1985;Brunton et al., “Goodman and Gilman's The Pharmacological Basis ofTherapeutics,” McGraw-Hill, 2005; University of the Sciences inPhiladelphia (eds.), “Remington: The Science and Practice of Pharmacy,”Lippincott Williams & Wilkins, 2005; and University of the Sciences inPhiladelphia (eds.), “Remington: The Principles of Pharmacy Practice,”Lippincott Williams & Wilkins, 2008.

Pharmaceutical compositions can be formulated by standard techniquesusing one or more physiologically acceptable carriers or excipients. Theformulations can contain a buffer and/or a preservative. The compoundsand their physiologically acceptable salts and solvates can beformulated for administration by any suitable route, including viainhalation, topically, nasally, orally, parenterally (e.g.,intravenously, intraperitoneally, intravesically or intrathecally) orrectally in a vehicle comprising one or more pharmaceutically acceptablecarriers, the proportion of which is determined by the solubility andchemical nature of the compound, chosen route of administration andstandard biological and pharmacological practices.

Additional routes of administration include, but are not limited to,transdermal, parenteral, intravenous, intra-arterial, subcutaneous,intramuscular, intracranial, intraorbital, ophthalmic, intraventricular,intracapsular, intraspinal, intracisternal, intraperitoneal,intracerebroventricular, intrathecal, intranasal, aerosol, bysuppositories, or by oral administration.

Pharmaceutical compositions can include effective amounts of one or morecompound(s) described herein together with, for example,pharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or other carriers. Such compositions caninclude diluents of various buffer content (e.g., TRIS or other amines,carbonates, phosphates, amino acids, for example, glycinamidehydrochloride (especially in the physiological pH range),N-glycylglycine, sodium or potassium phosphate (dibasic, tribasic),etc., TRIS-HCl or TRIS-acetate), pH and ionic strength; additives suchas detergents and solubilizing agents (e.g., surfactants such asPluronics, Tween 20, Tween 80, Polysorbate 80, Cremophor, polyols suchas polyethylene glycol, propylene glycol, etc.), anti-oxidants (e.g.,ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol,benzyl alcohol, parabens, etc.) and bulking substances (e.g., sugarssuch as sucrose, lactose, mannitol, polymers such aspolyvinylpyrrolidones or dextran, etc.); and/or incorporation of thematerial into particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronicacid may also be used.

Such compositions can be employed to influence the physical state,stability, rate of in vivo release, and rate of in vivo clearance of acompound described herein. See, e.g., Remington's PharmaceuticalSciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages1435-1712 which are hereby incorporated herein by reference. Thecompositions can, for example, be prepared in liquid form, or can be indried powder, such as lyophilized form. Particular methods ofadministering such compositions are described infra.

If a buffer is to be included in the formulations described herein, thebuffer can be selected from sodium acetate, sodium carbonate, citrate,glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogenphosphate, disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)-aminomethane, or mixtures thereof. The buffer canalso be glycylglycine, sodium dihydrogen phosphate, disodium hydrogenphosphate, and sodium phosphate or mixtures thereof. If apharmaceutically acceptable preservative is to be included in aformulation of one of the compounds described herein, the preservativecan be selected from phenol, m-cresol, methyl p-hydroxybenzoate, propylp-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate,2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, ormixtures thereof. The preservative can also be phenol or m-cresol.

The terms “pharmaceutically acceptable” and “therapeutically acceptable”refer to molecular entities and compositions that are physiologicallytolerable and preferably do not typically produce an allergic or similaruntoward reaction, such as gastric upset, dizziness and the like, whenadministered to a subject (e.g., a human).

In some embodiments, the compounds described herein can be administeredby any suitable route, including, but not limited to, via inhalation,topically, nasally, orally, parenterally (e.g., intravenously,intraperitoneally, intravesically or intrathecally) or rectally in avehicle comprising one or more pharmaceutically acceptable carriers, theproportion of which is determined by the solubility and chemical natureof the compound, chosen route of administration and standard practice.Administration of the compounds described herein can be carried outusing any method known in the art. For example, administration may betransdermal, parenteral, intravenous, intra-arterial, subcutaneous,intramuscular, intracranial, intraorbital, ophthalmic, intraventricular,intracapsular, intraspinal, intracisternal, intraperitoneal,intracerebroventricular, intrathecal, intranasal, aerosol, bysuppositories, or by oral administration. A pharmaceutical compositionof the compounds described herein can be for administration forinjection, or for oral, pulmonary, nasal, transdermal, or ocularadministration.

Exemplary formulations include, but are not limited to, those suitablefor parenteral administration, e.g., intrapulmonary, intravenous,intra-arterial, intra-ocular, intra-cranial, sub-meningial, orsubcutaneous administration, including formulations encapsulated inmicelles, liposomes or drug-release capsules (active agents incorporatedwithin a biocompatible coating designed for slow-release); ingestibleformulations; formulations for topical use, such as eye drops, creams,ointments and gels; and other formulations such as inhalants, aerosolsand sprays. The dosage of the compositions of the disclosure will varyaccording to the extent and severity of the need for treatment, theactivity of the administered composition, the general health of thesubject, and other considerations well known to the skilled artisan.

In additional embodiments, the compositions described herein aredelivered locally. Localized delivery allows for the delivery of thecomposition non-systemically, thereby reducing the body burden of thecomposition as compared to systemic delivery. Such local delivery can beachieved, for example, through the use of various medically implanteddevices including, but not limited to, stents and catheters, or can beachieved by inhalation, injection or surgery. Methods for coating,implanting, embedding, and otherwise attaching desired agents to medicaldevices such as stents and catheters are established in the art andcontemplated herein.

For oral administration, the pharmaceutical composition of the compoundsdescribed herein can be formulated in unit dosage forms such as capsulesor tablets. The tablets or capsules can be prepared by conventionalmeans with pharmaceutically acceptable excipients, including bindingagents, for example, pregelatinised maize starch, polyvinylpyrrolidone,or hydroxypropyl methylcellulose; fillers, for example, lactose,microcrystalline cellulose, or calcium hydrogen phosphate; lubricants,for example, magnesium stearate, talc, or silica; disintegrants, forexample, potato starch or sodium starch glycolate; or wetting agents,for example, sodium lauryl sulphate.

Tablets can be coated by methods well known in the art. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups, or suspensions, or they can be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives, for example, suspending agents,for example, sorbitol syrup, cellulose derivatives, or hydrogenatededible fats; emulsifying agents, for example, lecithin or acacia;non-aqueous vehicles, for example, almond oil, oily esters, ethylalcohol, or fractionated vegetable oils; and preservatives, for example,methyl or propyl-p-hydroxybenzoates or sorbic acid. The preparations canalso contain buffer salts, flavoring, coloring, and/or sweetening agentsas appropriate. If desired, preparations for oral administration can besuitably formulated to give controlled release of the active compound.

The compounds described herein can also include derivatives referred toas prodrugs, which can be prepared by modifying functional groupspresent in the compounds in such a way that the modifications areremoved (e.g., cleaved), either in routine manipulation or in vivo, toregenerate the parent compounds. Examples of prodrugs include compoundsof the invention as described herein that contain one or more molecularmoieties appended to a hydroxyl, amino, sulfhydryl, or carboxyl group ofthe compound, and that when administered to a patient, are cleaved invivo to form the free hydroxyl, amino, sulfhydryl, or carboxyl group,respectively. Examples of prodrugs include, but are not limited to,acetate, formate and benzoate derivatives of alcohol and aminefunctional groups in the compounds described herein. Preparation and useof prodrugs is discussed, for example, in T. Higuchi et al., “Pro-drugsas Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, andin “Bioreversible Carriers in Drug Design,” ed. Edward B. Roche,American Pharmaceutical Association and Pergamon Press, 1987, both ofwhich are incorporated herein by reference in their entireties.

Dosages

The compounds described herein can be administered to a subject attherapeutically effective doses to prevent, treat, or control one ormore diseases and disorders mediated, in whole or in part, by anOR-ligand interaction. The compounds can also be administered, eitheralone or in combination with other substances, for pain relief,induction of analgesia, reduction of nociception and/or to potentiatethe effect of an endogenous or exogenous opioid. Pharmaceuticalcompositions comprising one or more of compounds described herein can beadministered to a patient in an amount sufficient to elicit an effectiveprotective, therapeutic or analgesic response in a subject. An amountadequate to accomplish any of these is defined as a “therapeuticallyeffective dose.” A therapeutically effective dose is determined by theefficacy of the particular compound employed and the condition of thesubject, as well as the body weight or surface area of the region to betreated. The size of the dose can also be influenced by the existence,nature, and extent of any adverse effects that accompany theadministration of a particular compound or vector in a particularsubject.

Toxicity and therapeutic efficacy of compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, for example, by determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀ (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index and can be expressed as the ratio, LD₅₀/ED₅₀.In some embodiments, compounds that exhibit large therapeutic indicesare used. While compounds that exhibit toxic side effects can be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue to minimize potential damage tonormal cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused to formulate a dosage range for use in humans. In some embodiments,the dosage of such compounds lies within a range of circulatingconcentrations that include the ED₅₀ and that exhibits little or notoxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration and other factors,including the condition of the subject. For any compound describedherein, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose can be formulated in animal models toachieve a circulating plasma concentration range that includes the IC₅₀(the concentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma can be measured, for example, by high performance liquidchromatography (HPLC). In general, the dose equivalent of a modulator isfrom about 1 ng/kg to 10 mg/kg for a typical subject. In certainembodiments, the dose level is between 10 ng/kg and 1 mg/kg; in otherembodiments, between 100 ng/kg and 0.1 mg/kg; in other embodiments,between 1 μg/kg and 10 μg/kg. In additional embodiments, the dose rangefor a compound as described herein is between 1-100 ng/kg, or 10-1,000ng/kg, or 0.1-10 μg/kg, or 10-100 μg/kg, or 0.1-1 mg/kg, or 1-10 mg/kg.

The amount and frequency of administration of the compounds describedherein and/or the pharmaceutically acceptable salts thereof is regulatedaccording to the judgment of the attending clinician considering suchfactors as age, condition and size of the subject as well as severity ofthe symptoms being treated. An ordinarily skilled physician orveterinarian can readily determine and prescribe an effective amount ofa compound suitable to prevent, counter or arrest the progress of thecondition. In general, it is contemplated that an effective amount wouldbe from 0.001 mg/kg to 10 mg/kg body weight, and in particular from 0.01mg/kg to 1 mg/kg body weight. It may be appropriate to administer therequired dose as two, three, four or more sub-doses at appropriateintervals throughout the day. Said sub-doses can be formulated as unitdosage forms, for example, containing 0.01 to 500 mg, and in particular0.1 mg to 200 mg of active ingredient per unit dosage form.

Medical Uses

The compositions described herein are useful for treating pain orpain-associated disorders such as, for example, immune dysfunction,inflammation, esophageal reflux, neurological conditions, psychiatricconditions, urological conditions, sexual dysfunction and reproductiveconditions. They are also useful as medicaments for drug and alcoholabuse, agents for treating gastritis and diarrhea, cardiovascular agentsand agents for the treatment of respiratory diseases and cough.

In some embodiments, methods of treating pain are provided. In someembodiments, one or more compounds described herein are administered toa subject to treat the pain. In some embodiments, the pain can bepost-operative pain. In some embodiments, the pain is caused by cancer.In additional embodiments, the pain is caused by chemotherapy(chemotherapy-induced neuropathic pain, CINP). In some embodiments, thepain is associated with inflammation (i.e., inflammatory pain). In someembodiments, the pain is neuropathic pain. In some embodiments, the painis caused by trauma, such as but not limited to, blunt force trauma. Inadditional embodiments, the pain can result from endocrine imbalances,such as those resulting, e.g., from diabetes.

Kits

Another aspect of the present disclosure relates to kits for carryingout the administration of MOR PAMs to a subject. In one embodiment, akit comprises a composition comprising one or more MOR PAM(s),formulated as appropriate (e.g., in a pharmaceutical carrier), in one ormore separate pharmaceutical preparations. Kits can also contain devicesfor administration of the composition(s) and/or instructions for use.

EXAMPLES Example 1: Receptor Activation Assays

The ability of compounds to stimulate OR mediated signaling can bemeasured using any assay known in the art to detect OR mediatedsignaling or OR activity, or the absence of such signaling or activity.“OR activity” refers to the ability of an OR to transduce a signal. Suchactivity can be measured, e.g., in a heterologous cell, by coupling anOR (or a chimeric OR) to a downstream effector such as adenylatecyclase.

The effects of MOR PAMs and MOR SAMs on receptor activity were assayedusing the PathHunter enzyme complementation assay technology (DiscoveRx,CA). A description of the technology employed is as follows.

Beta-Arrestin Pathway: The PathHunter β-arrestin assay monitors theactivation of a GPCR in a homogenous, non-imaging assay format using atechnology developed by DiscoveRx called Enzyme Fragment Complementation(EFC) with beta-galactosidase (β-Gal) as the functional reporter. Theenzyme is split into two inactive complementary portions (EA for EnzymeAcceptor and ED for Enzyme Donor) expressed as fusion proteins in thecell. EA is fused to β-Arrestin and ED is fused to the GPCR of interest.When the GPCR is activated and β-arrestin is recruited to the receptor,ED and EA complementation occurs, restoring β-gal activity, which ismeasured using chemiluminescent PathHunter Detection Reagents.

cAMP Secondary Messenger Pathway: A cell line expressing stable MOR thatsignals through cAMP is used to quantify the activity of ligands forthis secondary pathway. Hit Hunter® cAMP assays monitor the activationof a MOR via Gi and Gs secondary messenger signaling in a homogenous,non-imaging assay format EFC with β-gal as the functional reporter.

Example 2: Allosteric Activity of Selected Compounds

Identification of positive allosteric modulators was performed in thepresence of an EC₂₀ concentration (40 nM) of the MOR-specificorthosteric agonist endomorphin-1. In this manner an EC₅₀ for each testcompound, and percent maximal response for each test compound, weredetermined for both the β-arrestin and cAMP pathways. Though thepharmacological effects of a given signal bias can be hypothesized(Raehal et al. (2011) Pharmacol. Rev. 63:1001-1019), it is not possibleto know with certainty which bias ratio will be most advantageous in aclinical setting. Compounding this issue is evidence that clinicallyused mixed agonist/partial agonist drugs for the MOR have significantlydifferent ligand bias. Kenakin 2015a, supra. The information associatedwith the compounds detailed herein is the first large data set availablefor the prediction of ligand bias for an MOR PAM. Example concentrationresponse curves are given in FIGS. 1A-5B.

Based on these assays, the compounds can be divided into a number ofcategories, for example: (A) Relative ratio of EC₅₀ for β-arrestin vs.cAMP (greater than, equal, or less than), (B) Maximal response ofβ-arrestin efficacy (silent, partial or full efficacy), (C) Maximalresponse of the cAMP efficacy (silent, partial or full efficacy), (D)the relative ratio of (B) to (C) and (E) the absolute values of the EC₅₀values for β-arrestin vs cAMP amongst compounds (high, moderate, and lowpotency). Exemplary data are provided in Tables 3 and 4 above. CompoundNo. 14 (FIGS. 3A and 3B) is an example of a compound with dual silentallosteric modulation of MOR with respect to either β-arrestin or cAMPsignaling. Compound No. 44 (Table 8) (FIGS. 1A and 1B) is an example ofa moderately potent compound that would be expected to have equivalenteffects on β-arrestin recruitment and cAMP signaling. Compound No. 219(Table 16) (FIGS. 2A and 2B) is an example of a moderately potentcompound that would be expected to have preferential signaling via cAMPover β-arrestin. Compound No. 216 (Table 11) (FIGS. 4A and 4B) is anexample of a compound that displays high allosteric modulatorycapability for β-arrestin recruitment and silent allosteric activitywith respect to cAMP signaling. Compound No. 2 (Table 12) (FIGS. 5A and5B) is an example of a potent compound that would be expected to haveroughly equivalent signaling for β-arrestin and cAMP.

Example 3: Lack of Agonist Activity by Compounds

To demonstrate that the compounds detailed herein display a pure PAMmechanism and do not have residual agonist or antagonist activity towardthe MOR, five compounds displaying MOR PAM activity (see FIGS. 6A-6J)were tested for both agonist and antagonist activity against the MOR,using a β-arrestin recruitment assay with the mu opioid receptor subtypeOPRM1 as the assay target. Agonist activity of endomorphin-1 was used asa positive control. The results shown in Table 18 demonstrate that thesefive diverse compounds do not show agonist or antagonist activity towardthe MOR as assayed by β-arrestin recruitment. Similar results wereobtained when agonist and antagonist activity were assayed using cAMPsignaling as the readout.

TABLE 18 Lack of agonist or antagonist activity of certain compounds astested by effects on β-arrestin recruitment Compound Assay format EC₅₀(μM) IC₅₀ (μM) 108 agonist >25 108 antagonist >25 177 agonist >25 177antagonist >25 6 agonist >25 6 antagonist >25 110 agonist >25 110antagonist >25 109 agonist >25 109 antagonist >25 endomorphin-1 agonist0.04889

Example 4: Concentration Dependence

Concentration-response curves (CRCs) for EM1-mediated cAMP signaling andβ-arrestin recruitment were generated in the absence or presence ofvarying concentrations of compound No. 9 (Table 10). The full functionalallosteric model for response (Kenakin (2005) Nature Reviews DrugDiscovery 4:919-927; Price et al. (2005) Mol. Pharmacol. 68:1484-1495;Ehlert (2005) J. Pharmacol. Exp. Ther. 315:740-754) was applied to DRcurves for EM1-mediated responses for β-arrestin and cAMP. This modelestimates the equilibrium dissociation constant of the modulator(K_(B)), the cooperative effect of the modulator on agonist affinity (α)and cooperative effect of the modulator on agonist efficacy (β).

Compound 9 produced concentration-dependent and saturable leftwardshifts in the potency of EM1 in both β-arrestin (FIG. 7A) and cAMP (FIG.7B) assays. With respect to β-arrestin signaling, compound 9 increasedthe affinity (α) of EM1 by a factor of 9 and increased the efficacy (β)of EM1 by a factor of 3; resulting in an effective potency for PAMeffect with respect to the β-arrestin signal of 70.8 nM. With respect tocAMP signaling, compound 9 increased the affinity (α) of EM1 by a factorof 2 and increased the efficacy (β) of EM1 by a factor of 1.8; resultingin an effective potency for PAM effect with respect to cAMP signaling of44 nM.

Example 5: Receptor Specificity

Allosteric ligands have the potential to exhibit greater selectivitybetween subtypes of GPCRs in the same family compared with orthostericligands. This effect has been demonstrated for some GPCRs includingmetabotropic glutamate receptors, adenosine receptors and muscarinicreceptors. Birdsall, supra; Conn et al., supra; Gao et al., supra;Gasparini et al. (2002) Curr. Opin. Pharmacol. 2:43-49. It has beenhypothesized that this selectivity arises from the evolutionaryconstraint placed on the orthosteric site between closely relatedreceptor subtypes that bind the same endogenous ligand. This proposedevolutionary constraint may or may not be present with respect toallosteric binding sites.

To test whether the MOR PAMs disclosed herein have effects on opioidreceptors other than the MOR, compound 60 was examined in β-arrestinrecruitment assays using U20S Path Hunter® cells expressing eitherPK-tagged delta opioid receptors (U20S-DOR1) or PK-tagged kappa opioidreceptors (U20S-KOR1). At concentrations up to 25 M, compound 60 had nosignificant positive allosteric effect on DOR signaling in the presenceof an EC₂₀ concentration (0.8 nM) of the delta opioid receptor agonist[D-Ala2, D-Leu5]-Enkephalin (DADLE) (FIG. 8A). The agonist activity ofDADLE on the DOR is shown in FIG. 8B for comparison.

Similarly, at concentrations up to 25 μM, compound 60 had no significantpositive allosteric effect on KOR signaling in the presence of an EC₂₀concentration (0.8 nM) of the Kappa opioid receptor agonist Dynorphin A(FIG. 9A). The agonist activity of Dynorphin A on the KOR is shown inFIG. 9B for comparison.

These results indicate that the effects of these ligands are mediatedthrough activation of mu-opioid receptors and that the compounds areselective for mu, over delta and kappa, opioid receptors.

Example 6: Probe Specificity

Opioid receptor tolerance, and eventual dependence, is thought to resultfrom prolonged exposure to opiates. This results in changes in cellfunction leading to the requirement for increased doses of agonist tomediate the same analgesic effect. This being the case, one therapeuticutility of the allosteric modulators disclosed herein is their abilityto modulate the activity of non-endogenous agonists. Using thisapproach, a lower dose of a given exogenous opioid (e.g., oxycodone,fentanyl), in the presence of a MOR PAM, will provide an analgesiceffect that is equivalent to that obtained by the use of a higherconcentration of the same probe in the absence of the MOR PAM. Thisallows use of a lower dose of opioid to achieve the desired therapeuticeffect; thereby affording a higher therapeutic index for patients in theclinical setting. By allowing the use of lower doses of opioid;therapeutic use of an opioid/MOR PAM combination is likely to retard oreliminate the development of tolerance, as well as reducinggastrointestinal distress, respiratory depression and other undesiredeffects of opioid use.

To test the specificity of MOR PAMs with respect to differentnon-endogenous MOR ligands (“probe specificity”), concentration responsecurves (CRCs) to identify the maximal response and EC₅₀ concentrationsfor the β-arrestin and cAMP responses of the OPRM1 receptor to fentanyl,morphine and oxycodone were performed and compared with the responses toEM1. The results are provided in Table 19.

TABLE 19 Signal-dependent EC₅₀ and maximum response determinations fordifferent MOR ligands Probe Assay EC₅₀ (μM) % Max. Resp. EM-1 β-arrestin0.066471 95.649 EM-1 cAMP 0.0017686 109.9 fentanyl β-arrestin 0.063364102.19 fentanyl cAMP 0.0029302 103.15 morphine β-arrestin 0.34612 104.65morphine cAMP 0.052858 109.28 oxycodone β-arrestin 2.3956 102.03oxycodone cAMP 0.078278 100.62

The data in Table 19 was used to determine EC₂₀ concentration offentanyl, morphine, oxycodone and EM1 for activation of the MOR, asmeasured by β-arrestin recruitment and adenylyl cyclase inhibition. CRCsfor various test compounds using EC₂₀ concentrations of fentanyl,morphine and oxycodone as the agonists were then performed and comparedto the results when EM1 was used as agonist. Representative data areshown in Tables 20-23.

TABLE 20 Allosteric effects of compounds on EC₂₀ morphine agonism of MORCompound Assay RC₅₀ (μM) Max. Response 6 β-arrestin 8.25 421 6 cAMP 2.9693 2 β-arrestin 9.59 156 2 cAMP >25 26 123 β-arrestin 14.09 59 123cAMP >25 0 8 β-arrestin 7.11 487 8 cAMP 2.00 44 9 β-arrestin 10.65 456 9cAMP 2.95 38

TABLE 21 Allosteric effects of compounds on EC₂₀ Fentanyl agonism of MORCompound Assay RC₅₀ (μM) Max. Response 6 β-arrestin 5.79 179 6 cAMP 2.0195 2 β-arrestin >25 19 2 cAMP >25 0 123 β-arrestin >25 0 123 cAMP >25 08 β-arrestin 14.25 146 8 cAMP >25 6 9 β-arrestin 13.57 83 9 cAMP >25 0

TABLE 22 Allosteric effects of compounds on EC₂₀ Oxycodone agonism ofMOR Compound Assay RC₅₀ (μM) Max. Response 6 β-arrestin 4.83 384 6 cAMP0.32 90 2 β-arrestin 13.56 126 2 cAMP >25 5 123 β-arrestin >25 17 123cAMP >25 0 8 β-arrestin 12.94 343 8 cAMP 1.91 56 9 β-arrestin 11.09 2969 cAMP 3.18 40

TABLE 23 Allosteric effects of compounds on EC₂₀ EM-1 agonism of MORCompound Assay RC₅₀ (μM) Max. Response 6 β-arrestin 2.52 173 6 cAMP 0.5093 2 β-arrestin 0.36 143 2 cAMP 0.57 81 123 β-arrestin 1.02 190 123 cAMP0.59 43 8 β-arrestin 0.33 213 8 cAMP 0.84 76 9 β-arrestin 0.36 210 9cAMP 0.59 75

As shown in Tables 21 and 23, most compounds displayed high selectivityfor the endogenous peptide EM1 over the non-endogenous synthetic opioidfentanyl as determined by either β-arrestin or cAMP readouts. Ofparticular interest is the probe dependency observed when comparing theeffects of identical allosteric modulators on the non-endogenousopium-based ligands morphine and oxycodone (Tables 20 and 22). Thesedata provide support for the proposition that the PAMs disclosed hereincan be used for the selective activation of the MOR using a variety ofnon-endogenous ligands for therapeutic use. The results provided inTables 20-23 provide direct evidence for ligand-dependent probe specificPAM activity; and thus provide a basis for the use of lower doses ofopioids, in combination with a probe-dependent PAM, to discriminatebetween the therapeutic analgesic properties of opioids, and theirtolerance and dependence liabilities. Moreover, the high level ofspecificity displayed for the endogenous OR agonist EM1 over thenon-endogenous probes indicates a low potential for abuse ofEM1-specific MOR PAMs.

Example 7: Selective Signaling Bias

The ability of the MOR PAMs disclosed herein to provide positiveallosteric modulation of the activity of exogenous opioids indicatesthat MOR PAMs can be used, in combination with exogenous opioids, toprovide analgesia with a reduced probability of the development oftolerance. An additional benefit would be realized if MOR PAMs also haddifferential effects of the various downstream processes induced byreceptor activation; some of which contribute to the side effects ofopioid use. Therefore, MOR PAMs that are able to bias the response to anorthosteric agonist away from signaling pathways that mediate tolerance,dependence and other unwanted effects, in favor of signaling pathwaysthat mediate a therapeutic response, would be desirable.

Accordingly, concentration-response curves (CRCs) for oxycodone-mediatedrecruitment of β-arrestin and oxycodone-mediated adenylyl cyclaseinhibition were generated in the absence or presence of varyingconcentrations of compound 6. The full functional allosteric model forresponse was applied to analyze the results. Kenakin (2005), supra;Price et al., supra; Ehlert, supra. This model estimates the equilibriumdissociation constant of the modulator (K_(B)), the cooperative effectof the modulator on agonist affinity (α) and cooperative effect of themodulator on agonist efficacy (β). Compound 6 producedconcentration-dependent and saturable leftward shifts in the potency ofoxycodone for both β-arrestin recruitment (FIG. 10A) and adenylylcyclase inhibition (FIG. 10B). With respect to the (β-arrestin signalingbias, Compound 6 increased the affinity (α) of oxycodone by a factor of2 and increased the efficacy (β) of oxycodone by a factor of 80. Withrespect to the cAMP signaling bias, compound 6 increased the affinity(α) of oxycodone by a factor of 4 and increased the efficacy (β) ofoxycodone by a factor of 4. This results in an effective potency for PAMeffect with respect to the cAMP signal of 234 nM.

Example 8: Pharmacokinetics

To be useful as a therapeutic, a MOR PAM must not only be able toactivate endogenous and non-endogenous orthosteric ligands of the MOR,it must also persist in the body in concentrations sufficient to exertits allosteric effect. To test intracorporeal persistence, murine modelswere chosen, based on the preponderance of data available linkingendogenous opioids to analgesic mechanisms. Compound 9 (Table 10) wasformulated for dosing in CD-1 mice. Two mice per group were tested andeach group was administered 15 mg/kg of the compound. Concentrations ofthe compound were measured by mass spectrometry of blood samples.Compound 9 was found to have exposures and an half-life appropriate forin vivo studies using either intraparential or subcutaneous dosing (FIG.11A).

Given that opioid receptors are highly concentrated in the dorsal rootganglion (DRG) and brain (Martin-Schild et al., supra), terminalpharmacokinetic studies were performed to verify the concentration inthe plasma, cerebrospinal fluid and brain. FIG. 11B shows that compound9 persists in brain and plasma, at therapeutically relevantconcentrations, one hour after either subcutaneous or intraperitonealadministration. These results indicate, for the first time, that a MORPAM can persist at EC₅₀ concentrations within a live animal, therebyjustifying further in vivo studies. (See Table 23, which shows that, forEM1-mediated MOR agonism, the RC₅₀ of compound 9 for β-arrestinrecruitment is 360 nM and the RC₅₀ of compound 9 for adenylyl cyclaseinhibition is 590 nM.)

Concentrations of subcutaneously injected compound 9 in spinal tissue(where the DRG are located) were also assessed. Compound 9 wasformulated for subcutaneous dosing in ICR rats and was found to exhibitgood exposures in spinal tissue (which contains the DRG) and brain,indicating coverage of the EC₅₀ in both relevant tissues (FIG. 11C).

Example 9: Augmentation of Anti-Nociceptive Effect of EM1 by a MOR PAM

An acute pain model was used to determine the analgesic effect of a MORPAM administered by itself (i.e., in the absence of exogenous opioid).This approach avoids the complexity of matching the pharmacokineticprofile of endomorphin release in response to a pain-inducing insultwith a (1) particular MOR PAM (to account for basal endomorphinanalgesis) and (2) the measurement window of the study, as would berequired for a chronic pain study. Accordingly, an acute pain model, inwhich the pharmacodynamic measurement was performed directly after theinsult, was employed. This was expected to isolate the low basal levelsof EM1 analgesis that existed prior to the insult, thereby eliminatingthe contribution of the time course of release of endogenous opioids inresponse to the insult. Mousa et al., supra.

The warm water tail-flick assay was chosen as the acute pain model forthe reasons described above. Exogenously applied EM1 has been shown todisplay potent analgesic effects in this model. Przewlocka et al.,(1999), supra. First, minimum and sub-efficacious dose of EM1, appliedvia intrathecal injection to rats, were identified. The values obtainedfor the minimum (3 ug/kg) and sub-efficacious dose of EM1 (1 ug/kg) arein agreement with those found in the literature. Horvath (2000), supra.In addition, the time required for maximal response to an intrathecalinjection of EM1 to be observed, as well as the time required for areturn to basal levels of antinociception, were determined and likewisefound to be in agreement with those found in the literature.

To verify the lack of antinociceptive effects of compound 9 when onlybasal EM1 is present, subcutaneous injections (15 mg/kg) of the compoundwere administered to CD1 mice (n=10) and tail-flick latency (TFL) wasassayed 30 minutes after dosing. No antinociceptive effects wereobserved 30 minutes after dosing (FIG. 12A).

In view of its lack of antinociceptive activity on its own, compound 9was then tested for in vivo MOR-PAM effects in conjunction withexogenously provided EM1 in ICR rats. For these experiments, the delayin tail flick induced by EM1 alone was compared to the delay in tailflick when compound 9 was dosed in ICR-rats in combination withintrathecal administration of minimum and sub-efficacious doses of EM1.Data for the time course antinociceptive effects of varying doses ofintrathecally administered EM1 is shown in FIG. 12B. Data for theeffects of two doses of compound 9 on the time course antinociceptiveeffects of a minimally efficacious dose (MED) of EM1 (3 μg) is shown inFIG. 12C. It can be seen that compound 9 potentiates the effect ofsuboptimal doses of EM-1 in the tail-flick assay; demonstrating that theantinociceptive effects of a minimally efficacious dose of EM1 can bemodulated in a dose dependent manner by a small molecule MOR-PAM. Thismodulation is observed as (a) a shortening of the time required forendomorphin to demonstrate antinocicepive effects, (b) an increase inmaximal response observed, and (c) a lengthening of the duration of theresponse. These effects might be due to any of the following:T_(on)[Endomorphin], K_(Elim)[Endomorphin], T_(off)[Endomorphin],K_(I)[Endomorphin], and possibly additional mechanisms.

An illuminating effect was observed at 80 min after administration, whenthe EM1 response was waning. Specifically, compound 9 demonstrated aβ-effect on endomorphin efficacy (i.e., an increase in maximalresponse), as was observed in vitro (see FIGS. 7A and 7B). The in vivoeffects of compound 9 can be modelled by the in vitro parametersobtained for this PAM (FIGS. 7A and 7B) with considerable similarity.Specifically, while the in vitro β-arrestin endomorphin responsesmodeled in FIG. 7A yielded an αβ product of 27, the in vivo effects(FIG. 12C) were fit with an ca3 product of 31.5.

The effect of compound 9 on the antinociceptive activity of asub-efficaceous dose of EM-1 was also assessed. Data for the effects oftwo doses of compound 9 on the time course antinociceptive effects of asub-efficacious dose of EM1 (1 μg) is shown in FIG. 12D. The resultsshow that compound 9 also potentiates the effect of sub-efficaceousdoses of the opioid. These results demonstrate that the anti-nociceptiveeffects of a sub-efficacious dose of endomorphin-1 can be modulated, ina dose dependent manner, by a small molecule MOR PAM. This modulation isobserved as (a) a shortening of the time required for endomorphin-1 todemonstrate its anti-nociceptive effects, (b) an increase in maximalresponse observed, and (c) an increase in the duration of the response.The ability to rescue a sub-therapeutic dose of the endogenous ligand ina dose- and time-dependent manner with a subtype selective MOR PAM hassignificant implications for the clinical viability of the use of MORPAMs in acute and chronic pain settings.

The positive allosteric effects of compound 9 on endomorphin activityare consistent with increased endomorphin efficacy (i.e., an increased βvalue) induced by compound 9, as was observed in vitro (FIGS. 7A and7B). This effect is valuable, as discussed previously, in terms ofincreasing low levels of response. However, the affinity effect (avalue) is also significant, since the overall affinity of all PAMsdepends on the co-binding ligand and also the magnitude of a. Kenakin(2012) Brit. J. Pharmacol. 165:1659-1669. The fact that α is >1 forcompound 9 indicates that it will bind the MOR with reasonably highaffinity in the brain in the presence of endomorphin. This is due to thereciprocal effect of allosteric energy, i.e. as compound 9 increases theaffinity of EM1, so too does EM1 increase the affinity of the receptorfor compound 9. The positive 1 effects of compound 9 may be especiallybeneficial as PAM-induced changes in efficacy have been shown to beuniquely powerful.

Example 10: Activity Switching

It has been observed that allosteric modulators of GPCRs can oftenexhibit “activity switching” within a chemical series: this occurs whenminor modifications to its chemical structure change a compound from aPAM to a negative (NAM) or silent (SAM) allosteric modulator. The lossof observed PAM efficacy that accompanies activity switching may be dueto loss of binding affinity, or functional switching from PAMs to NAMsor SAMs. Examples are provided in Table 24.

TABLE 24 Activity switching by MOR PAMs* β-arrestin β-arrestin cAMP cAMPNo. EC₅₀ Max Response EC₅₀ Max Resp. 18 C F C D 55 C F C D 57 C F C E 60C F C D 63 C F C E 71 C F C D 72 B F A D 73 C F C D 79 C F C D 83 C D CD Legend: The first column provides the compound number (identifiedelsewhere herein) of the compound tested. The second column providesEC₅₀ values for β-arrestin recruitment; the third column providesmaximal response values for β-arrestin recruitment; the fourth columnprovides EC₅₀ values for adenylyl cyclase inhibition; and the fifthcolumn provides maximal response values for adenylyl cyclase inhibition.EC₅₀ values are coded as follows: A represents an EC₅₀ < 700 nM; Brepresents an EC₅₀ between 700 nM and 2.1 μM; and C represents an EC50 > 2.1 μM. Maximal response values are coded as follows: D representsa maximal response of < 40%; E represents a maximal response of 40-60%;and F represents a maximal response of >60%.

Example 11: Activity of EM1-Specific MOR PAMs on EM2 Activity

As EM1 and EM2 have very similar structures and activities, but havedifferential concentrations in relevant tissues, it is necessary toassess the activity of EM1-specific MOR PAMS with respect to EM2activity. Concentration response curves (CRCs) for three exemplarycompounds were obtained to identify the maximal response and EC₂₀concentrations for the β-arrestin and cAMP responses to EM1 (see Table23 above). These assays were then used to assess the effect of thesethree compounds on EM2 activity. This data is shown in Table 25. As canbe seen, the compounds retain potency against EM2 and have higher signalbias compared to EM1 (cf Table 23, above). Based on these results, thedirect correlation between the intensity of EM2 staining and theetiology of CCI-induced neuropathic pain described herein (see, e.g.,Example 13) can be modulated using an EM2 MOR PAM.

TABLE 25 Allosteric effects of compounds on EC₂₀ EM2 agonism of MORCompound Assay RC₅₀ (μM) Max. Response 6 β-arrestin 3.34 117 6 cAMP0.173 91 2 β-arrestin 0.61 97 2 cAMP 0.10 83 9 β-arrestin 0.78 138 9cAMP 0.14 80

Example 12: Inflammatory Pain

It is known that, upon inflammatory insult, the body traffics MORs fromthe dorsal root ganglia (DRG) to the site of injury. (Stein & Machelska,supra; (Stein et al. (1993) Lancet 342:321-324. Concomitant with thisrelocalization of MORs, trafficking of endogenous opioid peptides isthought to occur, possibly via leukocytes, in a temporally- andspatially-dependent fashion. (Przewlocki et al. (1992), supra; Rittneret al., supra; Stein et al. (1990) Proc. Natl. Acad. Sci. USA87:5953-5939; Martin-Schild et al., supra. Some of these leukocytes(CD45⁺3E7) have been shown to specifically traffic EM1 and EM2, and areupregulated in inflamed tissue compared to non-inflamed tissue. Mousa etal., supra.

To test the efficacy of MOR-PAMs for relief of inflammatory pain in amurine model, 50-150 μl of Freund's complete adjuvant is introduced intothe paw by intraplantar injection; inducing swelling and mechanicalhyperalgesia. Subsequent to the establishment of inflammation, 15 mg/kgof a MOR PAM (e.g., compound 9) is introduced into the animal bysubcutaneous injection. Prior to administration of the MOR PAM and atvarious time points thereafter, pain levels are assessed, e.g., bywithdrawal of the paw from mechanical pressure, using, for example, avon Frey apparatus or a Randall & Selitto apparatus. See, e.g., Martinovet al. (2013) J. Vis. Exp. 82:51212. After administration of the MORPAM, the pain response is reduced, and is further reduced at succeedingtime points.

It has been shown that in an inflammatory setting, additional exogenousstress can release EM1. This has been shown to result in a switchingfrom hyper to hypo algesia in the ipsilateral vs. contralateral paws.Rittner et al., supra. Accordingly, in further tests, after induction ofinflammation the rodent is subjected to stress to cause a release ofsuprabasal EM1 and is administered a MOR PAM (e.g., compound 9). Underthese conditions, hypoalgesia is amplified and occurs at lowerendogenous stress levels.

In additional experiments, a MOR PAM such as compound 9 is administeredprior to mechanical hyperalgesia and pain response is assessed atvarious times after induction of mechanical hyperalgesia. Pain responseis compared to an animal that has been subjected to mechanicalhyperalgesia but has not received a prior administration of a MOR PAM.At all times after induction of mechanical hyperalgesia, the animal thatreceived the MOR PAM shows a lower pain response.

Example 13: Neuropathic Pain

Pain arising from peripheral nerve injury is a severe disability and hasprofound societal impacts. Neuropathic pain (NP) is poorly treatedclinically and is an unmet medical need of great societal importance.Williams & Christo (2009) “Pharmacological and interventional treatmentsfor neuropathic pain.” In M. Dobretsov, & J. Zhang, Mechanisms of Painin Peripheral Neuropathy (pp. 295-375). Kerala, India: ResearchSignpost.

It has been shown in animal models of chronic constrictive injury (CCI)of nerves that the recruitment of opioid-containing leukocytes as wellas an increase in MOR density occurs at the site of injury. Vasudeva etal. (2014) PLoS One 9:e90589; Truong et al., supra; Stein & Machelska,supra. Some of the leukocytes that are recruited in response to CCI wereshown to traffic endogenous opioids. Celik et al. (2016) Brain, Behaviorand Immunity 57:227-242. Activation of these leukocytes upon exposure tocorticotrophin releasing factor (or other substances), is hypothesizedto cause a release of endogenous opioids, which ablates mechanicalhyperalgesia normally accompanied by neuropathic pain. Labuz et al.(2010) Brain, Behavior and Immunity 24:1045-1053; Celik et al. (2013)Brain, Behavior and Immunity 29:S2-S9. Furthermore, a direct correlationbetween the intensity of EM2 staining and the etiology of CCI inducedneuropathic pain has been shown (Smith et al. (2001) Neuroscience105:773-778. Additionally, in animal models of neuropathic pain, thereis substantial precedent that exogenously applied endogenous opioidshave significant effect (Horvath, supra; Przewlocka et al. (1999a),supra.

A number of animal model systems for neuropathic pain exist, including,for example, spinal crushing; Bennett, Xie or Setzler models; chronicconstriction injury (CCI); the sciatic nerve crush model; spinal nerveligation; and laser-induced sciatic nerve injury. See, for example,Jaggi et al. (2009) Fund. & Clin. Pharmacology 25:1-28. To test theefficacy of MOR-PAMs for relief of neuropathic pain, 15 mg/kg of a MORPAM (e.g., compound 9) is introduced into an animal (e.g., a mouse or arat) prior to, concurrent with, or subsequent to, induction ofneuropathic pain in one or more of these model systems. Administrationof the MOR PAM is by subcutaneous injection or orally (e.g., in theanimal's food or water). At various time points after administration ofthe MOR PAM, pain levels are assessed. After administration of the MORPAM, the pain response is reduced, and is further reduced at succeedingtime points.

In additional experiments, animals are subjected to procedures thatinduce neuropathic pain, and pain response in an animal receiving a MORPAM is compared to pain response in an animal that has not received aMOR PAM. At all time points, the animal that received the MOR PAM showsa lower pain response.

In further experiments, prophylactic addition of a MOR PAM upon nervedamage delays or ameliorates the onset or intensity of neuropathic pain.

Example 14: Chemotherapy-Induced Neuropathy

Pain arising from chemotherapy (chemotherapy induced neuropathic pain,CINP) is common (for example, it is estimated that 90 to 100% of femalesreceiving cisplatin or paclitaxel for ovarian cancer present symptoms ofCINP) and can be a disabling side effect of cancer treatment. In fact,the development of painful neuropathy is often a dose-limiting criterionfor oncologic medications. This often leaves the patient with thelimited options of either dose reduction or discontinuation ofpotentially curative or palliative chemotherapeutic agents. Kaley &DeAngelis (2009) Br. J. Haematology 145:3-14; Williams & Christo, supra.CINP incidence and severity are directly related to dose, number oftreatment cycles, duration of treatment, etc.

In animal models of CINP, it has been shown that the administration ofexogenous EM-1 and EM-2 at the spinal level results in a strongeranalgesic effect than that of morphine. Przewlocka et al. (1999a),supra; Grass et al. (2002) Neurosci. Letts. 324:197-200; Przewlocki etal. (1999b), supra. It has been found that there is a linear correlationbetween the spinal concentration of EM-2 and the degree of mechanicalhyperalgesia observed in an animal model of CINP. Yang et al., supra;(Chen et al. (2015) Neuroscience 286:151-161. It was further found thatdecreasing levels of EM2-like immunoreactivity was the consequence ofDPP4 upregulation, and that the concentration of EM-2, and thus themanifestation of CINP mediated mechanical hyperalgesia, can be halted bythe prophylactic addition of a DPP4 inhibitor.

Given the involvement of EM2 in relief of CINP, taken together with theMOR PAM effects on EM2 shown above (Example 11), MOR PAMS are used forthe relief of CINP. To this end, 15 mg/kg of a MOR PAM (e.g., compound9) is introduced into an animal (e.g., a mouse or a rat) prior to,concurrent with, or subsequent to, introduction of a chemotherapeuticagent, in one or more of the model systems described above.Administration of the MOR PAM is by subcutaneous injection or orally(e.g., in the animal's food or water). Control animals do not receive achemotherapeutic agent; additional controls receive a chemotherapeuticagent, but do not receive a MOR PAM. At various time points afteradministration of the MOR PAM, pain levels are assessed. Afteradministration of the MOR PAM, the pain response is reduced, and isfurther reduced at succeeding time points.

A possible contributory mechanism for CINP is the inability of theendogenous opioid system to manage the pain caused by the cytotoxicagent. As such, increasing the levels of endogenous opioids with DPP4inhibitors and/or increasing the potency and duration of the endogenousopioids may be a preventative method to delay the onset or intensity ofCINP. This could alter the dose limiting toxicity observed with manycytotoxic agents, thus relieving patients of the pain associated withthe administration of lifesaving medicine. The application of a MOR PAMto coincide with the physiologic peptidase upregulation that accompanieschemotherapy can ablate the mechanical hyperalgesia using the higherthan basal levels of the endogenous opioids. Additionally, the use of aMOR PAM in combination with the action of peptidase inhibitors (toincrease the concentration of EM1 and EM2), results in mechanicalhypoalgesia, delaying or ameliorating the onset or intensity ofneuropathic pain.

Example 15: Sexual Dysfunction

Just as hormone and neurotransmitter levels have been shown to haveeffects on behavior, similar effects have been demonstrated with theendogenous opioids. Thus, in addition to the amelioration of pain, thecompositions and methods disclosed herein can also be used to address avariety of additional conditions (e.g., depression, anxiety, etc.) thatresult from pain and/or stress; by using MOR PAMs to enhance the body'sability to modulate pain and/or stress via the action of endogenousligands.

One such condition is sexual dysfunction. Research has shown that opioidpeptides are involved in the copulatory behavior of male mammals,including humans. The regulatory effect of opioids on sexual behavior isdose-dependent and varies depending on the site of infusion. In humans,acute opioid administration results in an intense euphoria, but longterm opioid use is associated with a deterioration of sexual function.Deteriorated sexual function in humans and animals is associated withlong term opioid abuse, as evidence by but not limited to increasedintromission latency, a reduction in mounting frequency, and reductionin intromission frequency. Different lines of evidence suggest thatsexual behavior is a physiological stimulus that releases endogenousopioids with two possible effects: to facilitate subsequent sexualbehavior and to enhance the rewarding properties of mating andejaculation. It has been shown that exogenously administered EM1 hasdirect effects on the intromission latency, mount and intromissionfrequency in a murine model. Parra-Gamez et al. (2009) Physiology andBehavior 97:98-101. Though intensity of the ejaculatory events could notbe quantified, this evidence indicates that sexual behavior andejaculation can be influenced by the release of endogenous opioids. Thisadds to a body of evidence that EM1 is involved with the rewardingaspects associated with mating.

In light of the involvement of EM1 in male sexual behavior, MOR PAMs canbe used to treat certain types of male sexual dysfunction. To test theeffects of MOR-PAMs for relief of sexual dysfunction, 15 mg/kg of a MORPAM (e.g., compound 9) is introduced into a male or female animal (e.g.,a mouse or a rat) prior to, concurrent with, or subsequent to,introduction to a sexually active partner of the opposite gender.Administration of the MOR PAM is by subcutaneous injection or orally(e.g., in the animal's food or water). At various time points afteradministration of the MOR PAM, intromission latency, mount andintromission frequency are assessed. Animals receiving the MOR PAM haveshorter intromission latency, higher mount frequencies and higherintromission frequencies than animals that did not receive a MOR PAM.

Example 16: Gastrointestinal Transit

Inhibition of gastrointestinal transit is a significant side effectassociated with opioid treatment of acute and chronic pain. Efforts togenerate analgesics that dissociate pain relief from side effects arecritical to the advancement of public health. Drug-related side effectsaffecting bowel function including, for example, constipation, abdominalconstriction and diarrhea, can cause a great deal of discomfort andpain.

The GI transit model is designed to identify compounds that produceeffects on bowel function and cause inhibition of gut transit. Theopioid comparator compound, morphine, is well known to inhibitgastrointestinal function. GI transit is an intestinal transport modelmeasuring the distance traveled through the GI tract by an orallyadministered charcoal bolus. Opioids, which are known to causeconstipation in the clinic, slow the transport of the charcoal bolus.

An experiment was performed to examine the effects of a MOR PAM compoundon the rate of GI transit in comparison to negative (vehicle) andpositive (morphine) controls. Sprague-Dawley rats were fasted for 18 hrsprior to the start of the study. Morphine (10 mg/kg in saline), compound9 (15, 30, 60 mg/kg in 5% DMSO: 10% cremophor EL: 85% H₂O) and vehicle(5% DMSO:10% cremophor EL: 85% H₂O) were administered subcutaneously 1hour prior to administration of the charcoal suspension. Charcoal andgum arabic were suspended in distilled water at a ratio of 10 grams ofcharcoal:2.5 grams of gum arabic:100 mL of water. The charcoalsuspension was administered by lavage to rats at a dose of 1 mL/100 g ofbody weight. Twenty minutes after charcoal administration rats weresacrificed and the small intestine was removed. The stomach was weighedbefore and after flushing with saline and the values recorded. Thedifference (stomach contents) was calculated. The small intestine wasuncoiled and the distance covered by the charcoal was measured andrecorded. Percent transit was calculated for each rat using the formula:(distance charcoal travelled/length of intestine)×100.

As shown in FIG. 13, GI transit was significantly decreased by morphine(10 mg/kg). In contrast, GI transit was unchanged followingadministration of compound 9. As shown in FIG. 14, stomach weight wasincreased after administration of morphine (10 mg/kg). In contrast,stomach weight was unchanged following administration of the MOR PAMcompound 9. These results provide evidence that a MOR PAM (at doses of15, 30, 60 mg/kg) does not exhibit opioid-like impairment of GI transit.

Example 17: Respiratory Depression

Respiratory depression is a significant side effect associated withopioid treatment of acute and chronic pain. Drug-related overdose deathsare often a function of the respiratory depression which occurs withopioid use. Efforts to generate analgesics that dissociate pain relieffrom side effects are critical to the advancement of public health.Pulse oximetry has been demonstrated to be a reliable non-invasivemethod for evaluating respiratory depression. Blood oxygen saturationlevels in awake rats are measured and compared, using morphine as apositive control.

An experiment was performed to examine the effects of a MOR PAM compoundon oxygen saturation levels in comparison to negative (vehicle) andpositive (morphine) controls. After baseline pulse oximetrymeasurements, Sprague-Dawley rats were dosed sucutaneously with morphine(10 mg/kg in saline), compound 9 (15, 30, 60 mg/kg in 5% DMSO:10%cremophor EL: 85% H₂O) or vehicle (5% DMSO:10% cremophor EL: 85% H₂O)and examined for 90 minutes. Blood oxygen saturation was assessed inawake rats with the MouseOx oximeter system (Starr Life Sciences,Holliston, Mass.). Up to 5 samples were measured every 5 minutes for 15minutes prior to dosing and 90 minutes after dosing. Each animal washabituated to the device over several days prior to the study.

As shown in FIG. 15, morphine (10 mg/kg) resulted in a significantreduction in oxygen saturation. In comparison, none of the dosesevaluated for the MOR PAM compound 9 resulted in a reduction of oxygensaturation compared to vehicle at any point during the 90 minuteevaluation. This indicates that the MOR PAM compound did not inducerespiratory depression that is typically associated with opioidcompounds such as morphine. This is the first demonstration that an MORPAM does not cause opioid-like respiratory depression.

1-20. (canceled)
 21. A method for treating a condition in a subject byadministering a positive allosteric modulator of the mu opioid receptor(a MOR-PAM) to the subject.
 22. The method of claim 21, wherein thecondition is selected from the group consisting of substance abuse,acute pain, chronic pain, inflammatory pain, neuropathic pain,chemotherapy-induced neuropathy, sexual dysfunction, abdominalconstruction, inhibited gut transit, constipation, respiratorydepression and allodynia.
 23. A method for modulating a processresulting from activation of the mu opioid receptor in a subject, themethod comprising administering a MOR-PAM to the subject.
 24. The methodof claim 23, wherein the process is recruitment of β-arrestin.
 25. Themethod of claim 23, wherein the process is inhibition of adenylatecyclase activity
 26. The method of claim 23, wherein the process isphosphorylation of ERK1/2.
 27. The method of claim 23, wherein theprocess is G-protein activation.
 28. A kit comprising a MOR-PAM.
 29. Thekit of claim 28, further comprising a pharmaceutical carrier.
 30. Thekit of claim 28, further comprising a device for administration of theMOR-PAM.
 31. The kit of claim 29, further comprising a device foradministration of the MOR-PAM.